RESUMEN
Myeloid cells are the first line of defence against pathogens. Mitochondrial apoptosis signalling is a crucial regulator of myeloid cell lifespan and modulates the function of myeloid cells. The anti-apoptotic protein BCL-2-family protein BCL2A1/A1/BFL-1 is strongly upregulated in inflammation in macrophages. We analysed the contribution of A1 to apoptosis regulation in a conditional system of in vitro differentiation of murine macrophages from immortalised progenitors. We disabled the expression of A1 by targeting all murine A1 isoforms in the genome. Specific inhibitors were used to inactivate other anti-apoptotic proteins. Macrophage progenitor survival mainly depended on the anti-apoptotic proteins MCL-1, BCL-XL and A1 but not BCL-2. Deletion of A1 on its own had little effect on progenitor cell survival but was sensitised to cell death induction when BCL-XL or MCL-1 was neutralised. In progenitors, A1 was required for survival in the presence of the inflammatory stimulus LPS. Differentiated macrophages were resistant to inhibition of single anti-apoptotic proteins, but A1 was required to protect macrophages against inhibition of either BCL-XL or MCL-1; BCL-2 only had a minor role in these cells. Cell death by neutralisation of anti-apoptotic proteins completely depended on BAX with a small contribution of BAK only in progenitors in the presence of LPS. A1 and NOXA appeared to stabilise each other at the posttranscriptional level suggesting direct binding. Co-immunoprecipitation experiments showed the binding of A1 to NOXA and BIM. Interaction between A1 and Noxa may indirectly prevent neutralisation and destabilization of MCL-1. Our findings suggest a unique role for A1 as a modulator of survival in the macrophage lineage in concert with MCL-1 and BCL-XL, especially in a pro-inflammatory environment.
Asunto(s)
Apoptosis , Diferenciación Celular , Supervivencia Celular , Macrófagos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2 , Proteína bcl-X , Animales , Proteína bcl-X/metabolismo , Macrófagos/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Lipopolisacáridos/farmacología , Células Mieloides/metabolismoRESUMEN
Proteasome inhibitors have been employed in the treatment of relapsed multiple myeloma and mantle cell lymphoma. The observed toxicity caused by proteasome inhibitors is a universal phenotype in numerous cancer cells with different sensitivity. In this study, we investigate the conserved mechanisms underlying the toxicity of the proteasome inhibitor bortezomib using gene editing approaches. Our findings utilizing different caspase knocking out cells reveal that bortezomib induces classic intrinsic apoptosis by activating caspase-9 and caspase-3/7, leading to pore-forming protein GSDME cleavage and subsequent lytic cell death or called secondary necrosis, a phenotype also observed in many apoptosis triggers like TNFα plus CHX, DTT and tunicamycin treatment in HeLa cells. Furthermore, through knocking out of nearly all BH3-only proteins including BIM, BAD, BID, BMF and PUMA, we demonstrate that NOXA is the sole BH3-only protein responsible for bortezomib-induced apoptosis. Of note, NOXA is well known for selectively binding to MCL-1 and A1, but our studies utilizing different BH3 mimetics as well as immunoprecipitation assays indicate that, except for the constitutive interaction of NOXA with MCL-1, the accumulation of NOXA after bortezomib treatment allows it to interact with BCL-XL, then simultaneous relieving suppression on apoptosis by both anti-apoptotic proteins BCL-XL and MCL-1. In addition, though bortezomib-induced significant ER stress and JNK activation were observed in the study, further genetic depletion experiments prove that bortezomib-induced apoptosis occurs independently of ER stress-related apoptosis factor CHOP and JNK. In summary, these results provide a solid conclusion about the critical role of NOXA in inactivation of BCL-XL except MCL-1 in bortezomib-induced apoptosis.
Asunto(s)
Apoptosis , Bortezomib , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Inhibidores de Proteasoma , Proteínas Proto-Oncogénicas c-bcl-2 , Proteína bcl-X , Humanos , Apoptosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Bortezomib/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Inhibidores de Proteasoma/farmacología , Factor de Transcripción CHOP/metabolismo , Factor de Transcripción CHOP/genética , Células HeLa , Sistema de Señalización de MAP Quinasas/efectos de los fármacosRESUMEN
Interactions between proteins and α-helical peptides have been the focus of drug discovery campaigns. However, the large interfaces formed between multiple turns of an α-helix and a binding protein represent a significant challenge to inhibitor discovery. Modified peptides featuring helix-stabilizing macrocycles have shown promise as inhibitors of these interactions. Here, we tested the ability of N-terminal to side-chain thioether-cyclized peptides to inhibit the α-helix binding protein Mcl-1, by screening a trillion-scale library. The enriched peptides were lariats featuring a small, four-amino-acid N-terminal macrocycle followed by a short linear sequence that resembled the natural α-helical Mcl-1 ligands. These "Heliats" (helical lariats) bound Mcl-1 with tens of nM affinity, and inhibited the interaction between Mcl-1 and a natural peptide ligand. Macrocyclization was found to stabilize α-helical structures and significantly contribute to affinity and potency. Yet, the 2nd and 3rd positions within the macrocycle were permissible to sequence variation, so that a minimal macrocyclic motif, of an N-acetylated d-phenylalanine at the 1st position thioether connected to a cysteine at the 4th, could be grafted into a range of peptides and stabilize helical conformations. We found that d-stereochemistry is more helix-stabilizing than l- at the 1st position in the motif, as the d-amino acid can utilize polyproline II torsional angles that allow for more optimal intrachain hydrogen bonding. This mixed stereochemistry macrocyclic N-cap is synthetically accessible, requiring only minor modifications to standard solid-phase peptide synthesis, and its compatibility with peptide screening can provide ready access to helix-focused peptide libraries for de novo inhibitor discovery.
Asunto(s)
Compuestos Macrocíclicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Péptidos , Estereoisomerismo , Péptidos/química , Péptidos/síntesis química , Péptidos/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Humanos , Conformación Proteica en Hélice alfa , Modelos MolecularesRESUMEN
MicroRNA hsa-miR-29 was connected to a number of malignancies. Its target genes are many, among them Mcl-1 that is expressed in three possible isoforms, one of which is anti-apoptotic and another one pro-apoptotic. Ratio of these two isoforms appears to affect cell response to external stimuli. We have demonstrated that miR-29b enhanced etoposide toxicity in HeLa cell line by modulating this ratio of Mcl-1 isoforms. However, it is not known whether the described miR-29 effect is common to various cancer types or even have the opposite effect. This represents a significant problem for possible future applications. In this report, we demonstrate that miR-29b affects toxicity of 60 µM etoposide in cell lines derived from selected malignancies. The mechanism, however, differs among the cell lines tested. Hep G2 cells demonstrated similar effect of miR-29b on etoposide toxicity as was described in HeLa cells, i.e. modulation of Mcl-1 expression. Target protein down-regulated by miR-29b resulting in enhanced etoposide toxicity in Caco-2 cells was, however, Bcl-2 protein. Moreover, H9c2, Hek-293 and ARPE-19 cell lines selected as a representatives of non-malignant cells, showed no effect of miR-29b on etoposide toxicity. Our data suggest that miR-29b could be a common enhancer of etoposide toxicity in malignant cells due to its modulation of Bcl family proteins.
Asunto(s)
Etopósido , MicroARNs , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Humanos , Etopósido/toxicidad , Etopósido/farmacología , MicroARNs/genética , MicroARNs/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células HeLa , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células HEK293 , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/toxicidad , Células Hep G2 , Células CACO-2RESUMEN
BACKGROUND: Cervical cancer ranks as one of the most prevalent malignancies among women worldwide and poses a significant threat to health and quality of life. MCL1 is an antiapoptotic protein closely linked to tumorigenesis, drug-resistance and poor prognosis in various cancers. Sanggenon C, a natural flavonoid derived from Morus albal., exhibits multiple activities, including anti-oxidant, anti-inflammatory, antivirus, and antitumor properties. However, the molecular mechanisms by which Sanggenon C exerts antitumor effects on in cervical cancer remain unclear. PURPOSE: To investigate the oncogenic role of MCL1 and elucidate the antitumor activity of Sanggenon C, along with its molecular mechanisms, in cervical cancer. METHODS: In vitro, the effects of Sanggenon C on proliferation, the cell cycle, apoptosis, and autophagy were explored. Transcriptome sequencing was employed to analyze critical genes and pathways. The expression of genes or proteins was evaluated via immunofluorescence, qRT-PCR, immunohistochemistry, and Western blotting. To identify targets of Sanggenon C, various techniques such as clinical database analysis, molecular docking, cellular thermal shift assays, co-immunoprecipitation, and ubiquitination assays were utilized. Additionally, Xenograft mouse models were established to further investigate Sanggenon C as a novel MCL1 inhibitor and its anti-tumor activity in vivo. RESULTS: Our investigation reveals that Sanggenon C effectively inhibits cervical cancer cell proliferation both in vitro and in vivo. Furthermore, Sanggenon C induces endoplasmic reticulum stress and triggers protective autophagy via activation of the ATF4-DDIT3-TRIB3-AKT-MTOR signaling axis. Furthermore, Sanggenon C specifically targets MCL1 to exert its antitumor effects by modulating MCL1 protein stability through SYVN1-mediated ubiquitination. Notably, MCL1 overexpression attenuates the Sanggenon C-induced decrease in cell viability and apoptosis. Our study further characterizes the role of MCL1 in cisplatin resistance and identifies MCL1 as a promising target for Sanggenon C, which effectively inhibits proliferation and induces apoptosis in cisplatin-resistant cervical cancer cells. Importantly, combining Sanggenon C with an autophagy inhibitor represents a promising strategy to enhance therapeutic outcomes in cisplatin-resistant cervical cancer cells. CONCLUSION: Our findings demonstrates that Sanggenon C induces endoplasmic reticulum stress and highlights the potential of targeting MCL1 to exploit vulnerabilities in drug-resistant cervical cancer cells. Sanggenon C emerges as a promising therapeutic agent against MCL1-driven adaptive chemoresistance through disruption of autophagy and endoplasmic reticulum stress in cervical cancer.
Asunto(s)
Autofagia , Resistencia a Antineoplásicos , Estrés del Retículo Endoplásmico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias del Cuello Uterino , Femenino , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/metabolismo , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Autofagia/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Ratones Desnudos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HeLa , Ratones Endogámicos BALB C , Ratones , Antineoplásicos Fitogénicos/farmacología , Simulación del Acoplamiento MolecularRESUMEN
Increasing the levels of antiapoptotic Bcl-2 proteins is an important way that cancer cells utilize to get out of apoptosis, underscoring their significance as promising targets for anticancer therapies. Lately, a primary compound 1 bearing thiazolidine-2,4-dione was discovered to exhibit comparable Mcl-1 inhibitory activity in comparison to WL-276. Herein, thirty-nine thiazolidine-2,4-dione analogs were yielded through incorporating different biphenyl moieties (R1), amino acid side chains (R2) and sulfonamides (R3) on 1. The findings indicated that certain compounds exhibited favorable inhibitory effects against Bcl-2/Mcl-1, while demonstrating limited or negligible binding affinity towards Bcl-xL. In particular, compounds 16 and 20 exhibited greater Bcl-2/Mcl-1 inhibition compared to AT-101, WL-276 and 1. Moreover, they demonstrated notable antiproliferative effects and significantly induced apoptosis in U937 cells. The western blot and co-immunoprecipitation assays confirmed that 20 could induce alterations in the expression of apoptosis-associated proteins to result in apoptosis through on-target Bcl-2 and Mcl-1 inhibition. In addition, 20 exhibited favorable stability profiles in both rat plasma and rat liver microsomes. In total, 20 could be used as a promising compound to discover Bcl-2/Mcl-1 dual inhibitors with favorable therapeutic properties.
Asunto(s)
Antineoplásicos , Apoptosis , Proliferación Celular , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2 , Tiazolidinedionas , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Estructura-Actividad , Proliferación Celular/efectos de los fármacos , Estructura Molecular , Apoptosis/efectos de los fármacos , Tiazolidinedionas/farmacología , Tiazolidinedionas/química , Tiazolidinedionas/síntesis química , Animales , Ratas , Desarrollo de MedicamentosRESUMEN
Infection of Rift Valley fever virus (RVFV), a highly pathogenic mosquito-borne zoonotic virus, triggers severe inflammatory pathogenesis but the underlying mechanism of inflammation activation is currently unclear. Here, we report that the non-structural protein NSs of RVFV triggers mitochondrial damage to activate the NLRP3 inflammasome leading to viral pathogenesis in vivo. It is found that the host transcription inhibition effect of NSs causes rapid down-regulation of myeloid cell leukemia-1(MCL-1), a pro-survival member of the Bcl-2 (B-cell lymphoma protein 2) protein family. MCL-1 down-regulation led to BAK activation in the mitochondria, which triggered mtROS production and release of oxidized mitochondrial DNA (ox-mtDNA) into the cytosol. Cytosolic ox-mtDNA binds and activates the NLRP3 inflammasome triggering NLRP3-GSDMD pyroptosis in RVFV infected cells. A NSs mutant virus (RVFV-NSsRM) that is compromised in inducing transcription inhibition did not trigger MCL-1 down-regulation nor NLRP3-GSDMD pyroptosis. RVFV infection of the Nlrp3-/- mouse model demonstrated that the RVFV-triggered NLRP3 pyroptosis contributed to RVFV inflammatory pathogenesis and fatal infection in vivo. Infection with the RVFV-NSsRM mutant virus similarly showed alleviated inflammatory pathogenesis and reduced fatality rate. Taken together, these results revealed a mechanism by which a virulence factor activates the mitochondrial MCL-1-BAK axis through inducing host transcription inhibition to trigger NLRP3-dependent inflammatory pathogenesis.
Asunto(s)
Mitocondrias , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteína con Dominio Pirina 3 de la Familia NLR , Piroptosis , Animales , Humanos , Ratones , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Inflamasomas/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/virología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Virus de la Fiebre del Valle del Rift , Proteínas no Estructurales ViralesRESUMEN
Mcl-1 is a main antiapoptotic protein in acute myeloid leukemia (AML) and is used as a target to develop inhibitors. Currently, potent Mcl-1 inhibitors primarily interact with the P2-P4 pockets of Mcl-1, but pharmacological modulation by targeting the P1 pocket is less explored. We designed a series of 1H-indole-2-carboxylic acid compounds as novel Mcl-1 inhibitors occupying the P1-P3 pockets and evaluated their Mcl-1 inhibition and apoptosis induction in AML cells. Two-dimensional 15N-HSQC spectroscopy indicated that 47 (Ki = 24 nM) bound to the BH3 binding groove, occupied the P1 pocket in Mcl-1, and formed interactions with Lys234 and Val249. 47 exhibited good microsomal stability and pharmacokinetic profiles, with low potential risk of cardiotoxicity. 47 inhibited tumor growth in HL-60 and THP-1 xenograft models with growth inhibition rate of 63.7% and 57.4%, respectively. Collectively, 47 represents a novel Mcl-1 inhibitor targeting the P1-P3 pockets with excellent antileukemia effects.
Asunto(s)
Antineoplásicos , Apoptosis , Indoles , Leucemia Mieloide Aguda , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Animales , Indoles/farmacología , Indoles/química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Estructura-Actividad , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Descubrimiento de Drogas , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Células HL-60 , Sitios de UniónRESUMEN
Myeloid cell leukemia 1 (Mcl-1) is a key regulator of the intrinsic apoptosis pathway. Overexpression of Mcl-1 is correlated with high tumor grade, poor survival, and both intrinsic and acquired resistance to cancer therapies. Herein, we disclose the structure-guided design of a small molecule Mcl-1 inhibitor, compound 26, that binds to Mcl-1 with subnanomolar affinity, inhibits growth in cell culture assays, and possesses low clearance in mouse and dog pharmacokinetic (PK) experiments. Evaluation of 26 as a single agent in Mcl-1 sensitive hematological and solid tumor xenograft models resulted in regressions. Co-treatment of Mcl-1-sensitive and Mcl-1 insensitive lung cancer derived xenografts with 26 and docetaxel or topotecan, respectively, resulted in an enhanced tumor response. These findings support the premise that pro-apoptotic priming of tumor cells by other therapies in combination with Mcl-1 inhibition may significantly expand the subset of cancers in which Mcl-1 inhibitors may prove beneficial.
Asunto(s)
Antineoplásicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Perros , Relación Estructura-Actividad , Femenino , Descubrimiento de Drogas , Taxoides/farmacología , Taxoides/farmacocinética , Taxoides/uso terapéutico , Taxoides/química , Docetaxel/farmacología , Docetaxel/uso terapéutico , Docetaxel/farmacocinética , Docetaxel/químicaRESUMEN
Dengue is a global health concern, and the host-viral interactions that regulate disease severity are largely unknown. Detrimental effects of neutrophils in this disease have been reported, but the precise mechanisms and functional properties of dengue-activated neutrophils are not fully characterised. Here, we measured the effects of dengue virus serotype 3 (DV3) on neutrophil lifespan and functions. We show that DV3 extends neutrophil survival with a significant proportion of cells surviving for 72 h post-incubation. These effects on neutrophil survival were greater than those observed by adding GM-CSF and TNF-α alone, but these cytokines enhanced survival induced by the virus. Enhanced reactive oxygen species (ROS) generation was observed following incubation with DV3 activation and this ROS production was enhanced by co-incubation with priming agents. In addition, DV triggered the enhanced IL-8 expression by the majority of neutrophils and a low percentage of cells were activated to express MCP-1 (CCL2). A low number of neutrophils showed increased co-expression of the migratory markers, CCR7 and CXCR4 which could promote their migration towards lymph nodes. DV3 significantly upregulated the BCL-XL gene at 3, 12, and 24 h, and the Mcl-1 gene at 12 h, following treatment. We also show that DV3 induces the Mcl-1 protein stabilization similar to GM-CSF. This report sheds new light on the mechanisms by which neutrophils may contribute to the pathology of dengue disease via delayed apoptosis and generation of pro-inflammatory molecules, and raises the possibility that dengue-activated neutrophils may play a role in activating cells of adaptive immunity.
Asunto(s)
Apoptosis , Virus del Dengue , Dengue , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neutrófilos , Especies Reactivas de Oxígeno , Virus del Dengue/fisiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Humanos , Dengue/inmunología , Dengue/virología , Especies Reactivas de Oxígeno/metabolismo , Serogrupo , Supervivencia CelularRESUMEN
Acute pancreatitis (AP) is an inflammatory disease of the pancreas and the main cause of hospital admissions for gastrointestinal diseases. Here, the work studied the circular RNA DTNB/microRNA-485-5p/MCL1 axis in AP and hoped to unravel the related mechanism. Caerulein exposure replicated an AP model in AR42J cells, and caerulein-mediated expression of circDTNB, miR-485-5p, and MCL1 was recorded. After exposure, cells were intervened with transfection plasmids and tested for LDH release, apoptosis, and inflammation. To determine the interwork of circDTNB, miR-485-5p, and MCL1, prediction results and verification experiments were conducted. Caerulein exposure reduced circDTNB and MCL1, while elevated miR-485-5p levels in AR42J cells. Upregulating circDTNB protected AR42J cells from caerulein-induced LDH cytotoxicity, apoptosis, and inflammation, but circDTNB upregulation-induced protections could be muffled by inhibiting MCL1. On the contrary, downregulating circDTNB further damaged AR42J cells under caerulein exposure, however, this phenomenon could be partially rescued after silencing miR-485-5p. miR-485-5p was mechanistically verified to be a target of circDTNB to mediate MCL1. Overall, the circDTNB/miR-485-5p/MCL1 axis protects inflammatory response and apoptosis in caerulein-exposed AR42J cells, promisingly identifying circDTNB as a novel molecule for AP treatment.
Asunto(s)
Apoptosis , Ceruletida , Inflamación , MicroARNs , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , ARN Circular , MicroARNs/genética , MicroARNs/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Animales , Ratas , Inflamación/genética , Pancreatitis/genética , Pancreatitis/metabolismo , Pancreatitis/inducido químicamente , Pancreatitis/patología , Línea CelularRESUMEN
Myeloid cell leukemia-1 (MCL-1), which belongs to the anti-apoptotic B cell lymphoma-2 family protein, is overexpressed in various cancers and is associated with cell immortality, malignant transformation, chemoresistance, and poor prognosis in humans. However, the significance of MCL-1 in canine mammary gland tumors (MGTs) remains unknown. This study aimed to examine MCL-1 expression in normal canine mammary glands and tumors and to assess its correlation with clinical and histologic variables. In total, 111 samples were examined, including 12 normal mammary gland tissues, 51 benign MGTs, and 48 malignant MGTs. Immunohistochemistry revealed that 53% of benign tumors and 75% of malignant tumors exhibited high MCL-1 expression, whereas only 8% of normal mammary glands exhibited high MCL-1 expression. High MCL-1 expression correlated with tumor malignancy (p < 0.001), large tumor size (> 3 cm) (p = 0.005), high Ki-67 expression (p = 0.046), and metastasis (p = 0.027). Survival curve analysis of dogs with malignant MGTs demonstrated a significant association between high MCL-1 expression and shorter median overall survival (p = 0.027) and progression-free survival (p = 0.014). Our study identified MCL-1 as a prognostic factor and potential therapeutic target in canine MGTs.
Asunto(s)
Enfermedades de los Perros , Neoplasias Mamarias Animales , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Animales , Perros , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Femenino , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Pronóstico , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/patología , Biomarcadores de Tumor/metabolismo , Inmunohistoquímica , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patologíaRESUMEN
Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Sinergismo Farmacológico , Neoplasias Pulmonares , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas Proto-Oncogénicas p21(ras) , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Animales , Línea Celular Tumoral , Ratones , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Transducción de Señal/efectos de los fármacos , Ciclina D1/metabolismo , Ciclina D1/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Femenino , Proteína 11 Similar a Bcl2/metabolismo , Proteína 11 Similar a Bcl2/genéticaRESUMEN
Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels. Moreover, we found that either selinexor treatment or DDX19A depletion effectively induced apoptosis of T-ALL lines expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.
Asunto(s)
ARN Helicasas DEAD-box , Hidrazinas , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , ARN Mensajero , Triazoles , Triazoles/farmacología , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Hidrazinas/farmacología , ARN Mensajero/genética , Leucemia/metabolismo , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucemia/patología , Apoptosis/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Antineoplásicos/farmacologíaRESUMEN
Promising outcomes have been reported in elder patients with acute myeloid leukemia (AML) using combined therapy of venetoclax (VEN) and azacytidine (AZA) in recent years. However, approximately one-third of patients appear to be refractory to this therapy. Vitamin K2 (VK2) shows apoptosis-inducing activity in AML cells, and daily oral VK2 (menaquinone-4, GlakayR) has been approved for patients with osteoporosis in Japan. We observed a high response rate to AZA plus VEN therapy, with no 8-week mortality in the newly diagnosed AML patients consuming daily VK2 in our hospital. The median age of the patients was 75.9 years (range 66-84) with high-risk features. Patients received AZA 75 mg/m2 on D1-7, VEN 400 mg on D1-28, and daily VK2 45 mg. The CR/CRi ratio was 94.7% (18/19), with a CR rate of 79%. Complete cytogenetic CR was achieved in 15 of 19 (79%) patients, and MRD negativity in 2 of 15 (13%) evaluable CR patients. Owing to the extremely high response rate in clinical settings, we further attempted to investigate the underlying mechanisms. The combination of VK2 and VEN synergistically induced apoptosis in all five AML cell lines tested. VK2, but not VEN, induced mitochondrial reactive oxygen species (ROS), leading to the transcriptional upregulation of NOXA, followed by MCL-1 repression. ROS scavengers repressed VK2 induced-NOXA expression and led to the cancellation of pronounced apoptosis and the downregulation of MCL-1 by VK2 plus VEN. Additionally, knockdown and knockout of NOXA resulted in abrogation of the MCL-1 repression as well as enhanced cytotoxicity by the two-drug combination, indicating that VK2 suppresses MCL-1 via ROS-mediated NOXA induction. These data suggest that the dual inhibition of BCL-2 by VEN and MCL-1 by VK2 is responsible for the remarkable clinical outcomes in our patients. Therefore, large-scale clinical trials are required.
Asunto(s)
Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes , Leucemia Mieloide Aguda , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2 , Sulfonamidas , Vitamina K 2 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Anciano , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Vitamina K 2/farmacología , Vitamina K 2/análogos & derivados , Vitamina K 2/uso terapéutico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Femenino , Masculino , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Sinergismo Farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Azacitidina/farmacología , Azacitidina/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.
Asunto(s)
Proteolisis , Humanos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Semivida , Doxorrubicina/farmacología , Doxorrubicina/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas/metabolismo , Proteínas/químicaRESUMEN
Triple negative breast cancers (TNBC) present a poor prognosis primarily due to their resistance to chemotherapy. This resistance is known to be associated with elevated expression of certain anti-apoptotic members within the proteins of the BCL-2 family (namely BCL-xL, MCL-1 and BCL-2). These regulate cell death by inhibiting pro-apoptotic protein activation through binding and sequestration and they can be selectively antagonized by BH3 mimetics. Yet the individual influences of BCL-xL, MCL-1, and BCL-2 on the sensitivity of TNBC cells to chemotherapy, and their regulation by cancer-associated fibroblasts (CAFs), major components of the tumor stroma and key contributors to therapy resistance remain to be delineated. Using gene editing or BH3 mimetics to inhibit anti-apoptotic BCL-2 family proteins in TNBC line MDA-MB-231, we show that BCL-xL and MCL-1 promote cancer cell survival through compensatory mechanisms. This cell line shows limited sensitivity to chemotherapy, in line with the clinical resistance observed in TNBC patients. We elucidate that BCL-xL plays a pivotal role in therapy response, as its depletion or pharmacological inhibition heightened chemotherapy effectiveness. Moreover, BCL-xL expression is associated with chemotherapy resistance in patient-derived tumoroids where its pharmacological inhibition enhances ex vivo response to chemotherapy. In a co-culture model of cancer cells and CAFs, we observe that even in a context where BCL-xL reduced expression renders cancer cells more susceptible to chemotherapy, those in contact with CAFs display reduced sensitivity to chemotherapy. Thus CAFs exert a profound pro-survival effect in breast cancer cells, even in a setting highly favoring cell death through combined chemotherapy and absence of the main actor of chemoresistance, BCL-xL.
Asunto(s)
Fibroblastos Asociados al Cáncer , Resistencia a Antineoplásicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neoplasias de la Mama Triple Negativas , Proteína bcl-X , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Línea Celular Tumoral , Femenino , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Resistencia a Antineoplásicos/genética , Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidoresRESUMEN
Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Péptidos , Unión Proteica , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Humanos , Péptidos/química , Péptidos/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Sitios de Unión , Secuencia de Aminoácidos , Proteína 11 Similar a Bcl2/metabolismo , Proteína 11 Similar a Bcl2/químicaRESUMEN
Lung cancer is the leading cause of cancer deaths, where the metastasis often causes chemodrug resistance and leads to recurrence after treatment. Desmethylclomipramine (DCMI), a bioactive metabolite of clomipramine, shows the therapeutic efficacy with antidepressive agency as well as potential cytostatic effects on lung cancer cells. Here, we demonstrated that DCMI effectively caused transforming growth factor (TGF)-ß1-mediated mesenchymal type of A549 cells to undergo mitochondrial death via myeloid cell leukemia-1 (Mcl-1) suppression and activation of truncated Bid (tBid). TGF-ß1 induced epithelial mesenchymal transition in A549 cells with the increase of fibronectin and decrease of E-cadherin, the activation of Akt/glycogen synthase kinase-3ß (GSK-ß)/Mcl-1 axis, and the hypo-responsiveness to cisplatin. DCMI initiated a dose-dependent cytotoxicity on TGF-ß1-mediated mesenchymal type of A549 cells through inactivating Akt/GSK-ß/Mcl-1 axis, in which mitochondria instability and caspase-9/3 activation also occurred concurrently. Pharmacological inhibition of caspase-8 and cathepsin B partly reversed tBid expression and mitochondrial damage to further attenuate DCMI-mediated cytotoxicity. Additionally, DCMI presented partial therapeutic effects in treating mesenchymal type of A549 tumor bearing nude mice through an acceleration of cancer cell death. Taken together, DCMI exerts antitumor effects via initiating the mechanisms of Akt/GSK-ß/Mcl-1 inactivation and cathepsin B/caspase-8-regulated mitochondrial death, which suggests its potential role in mesenchymal type of cancer cell therapy.
Asunto(s)
Transición Epitelial-Mesenquimal , Neoplasias Pulmonares , Mitocondrias , Animales , Humanos , Ratones , Células A549 , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Background: Gynostemma pentaphyllum (Thunb.) Makino, a well-known edible and medicinal plant, has anti-aging properties and is used to treataging-associated conditions such as diabetes, metabolic syndrome, and cardiovascular diseases. Gypenosides (GYPs) are the primary constituents of G. pentaphyllum. Increasing evidence indicates that GYPs are effective at preserving mitochondrial homeostasis and preventing heart failure (HF). This study aimed to uncover the cardioprotective mechanisms of GYPs related to mitochondrial regulation. Methods: The bioactive components in GYPs and the potential targets in treating HF were obtained and screened using the network pharmacology approach, followed by drug-disease target prediction and enrichment analyses. The pharmacological effects of GYPs in cardioprotection, mitochondrial function, mitochondrial quality control, and underlying mechanisms were further investigated in Doxorubicin (Dox)-stimulated H9c2 cardiomyocytes. Results: A total of 88 bioactive compounds of GYPs and their respective 71 drug-disease targets were identified. The hub targets covered MAPK, EGFR, PI3KCA, and Mcl-1. Enrichment analysis revealed that the pathways primarily contained PI3K/Akt, MAPK, and FoxO signalings, as well as calcium regulation, protein phosphorylation, apoptosis, and mitophagy process. In Dox-stimulated H9c2 rat cardiomyocytes, pretreatment with GYPs increased cell viability, enhanced cellular ATP content, restored basal oxygen consumption rate (OCR), and improved mitochondrial membrane potential (MMP). Furthermore, GYPs improved PINK1/parkin-mediated mitophagy without influencing mitochondrial fission/fusion proteins and the autophagic LC3 levels. Mechanistically, the phosphorylation of PI3K, Akt, GSK-3ß, and the protein level of Mcl-1 was upregulated by GYP treatment. Conclusion: Our findings reveal that GYPs exert cardioprotective effects by rescuing the defective mitophagy, and PI3K/Akt/GSK-3ß/Mcl-1 signaling is potentially involved in this process.