RESUMO
Oncogenic KRAS is considered a promising target for anti-cancer therapy. However, direct pharmacological strategies targeting KRAS-driven cancers remained unavailable. The prenyl-binding protein PDEδ, a transporter of KRAS, has been identified as a potential target for pharmacological inhibitor by selectively binding to its prenyl-binding pocket, impairing oncogenic KRAS signaling pathway. Here, we discovered a novel PDEδ inhibitor (E)-N'-((3-(tert-butyl)-2-hydroxy-6,7,8,9-tetrahydrodibenzo[b,dfuran-1-yl)methylene)-2,4-dihydroxybenzohydrazide(NHTD) by using a high-throughput docking-based virtual screening approach. In vitro and in vivo studies demonstrated that NHTD suppressed proliferation, induced apoptosis and inhibited oncogenic K-RAS signaling pathways by disrupting KRAS-PDEδ interaction in nonsmall cell lung cancer (NSCLC) harboring KRAS mutations. NHTD redistributed the localization of KRAS to endomembranes by targeting the prenyl-binding pocket of PDEδ and exhibited the suppression of abnormal KRAS function. Importantly, NHTD prevented tumor growth in xenograft and KRAS mutant mouse model, which presents an effective strategy targeting KRAS-driven cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/antagonistas & inibidores , Hidrazonas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Células A549 , Animais , Benzofuranos/farmacocinética , Benzofuranos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Feminino , Humanos , Hidrazonas/farmacocinética , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Nus , Células NIH 3T3 , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer deaths primarily due to chemoresistance. Somatic mutation of TP53 (36%) and epidermal growth factor receptor (EGFR; > 30%) are major contributors to cisplatin (CDDP) resistance. Substantial evidence suggests the elevated levels of reactive oxygen species (ROS) is a key determinant in cancer. The elevated ROS can affect the cellular responses to chemotherapeutic treatments. Although the role of EGFR in PI3K/Akt signaling cascade in NSCLC is extensively studied, the molecular link between EGFR and p53 and the role of ROS in pathogenesis of NSCLC are limitedly addressed. In this study, we investigated the role of p53 in regulation of ROS production and EGFR signaling, and the chemosensitivity of NSCLC. METHODS: In multiple NSCLC cell lines with varied p53 and EGFR status, we compared and examined the protein contents involved in EGFR-Akt-P53 signaling loop (EGFR, P-EGFR, Akt, P-Akt, p53, P-p53) by Western blot. Apoptosis was determined based on nuclear morphological assessment using Hoechst 33258 staining. Cellular ROS levels were measured by dichlorofluorescin diacetate (DCFDA) staining followed by flow cytometry analysis. RESULTS: We have demonstrated for the first time that activation of p53 sensitizes chemoresistant NSCLC cells to CDDP by down-regulating EGFR signaling pathway and promoting intracellular ROS production. Likewise, blocking EGFR/PI3K/AKT signaling with PI3K inhibitor elicited a similar response. Our findings suggest that CDDP-induced apoptosis in chemosensitive NSCLC cells involves p53 activation, leading to suppressed EGFR signaling and ROS production. In contrast, in chemoresistant NSCLC, activated Akt promotes EGFR signaling by the positive feedback loop and suppresses CDDP-induced ROS production and apoptosis. CONCLUSION: Collectively, our study reveals that the interaction of the p53 and Akt feedback loops determine the fate of NSCLC cells and their CDDP sensitivity.
RESUMO
microRNAs (miRNAs or miRs) are endogenous noncoding singlestranded RNA molecules that can regulate gene expression by targeting the 3'untranslated region and play an important role in many biological and pathological processes, such as inflammation and cancer. In this study, we found that miR20b was significantly increased in human nonsmall cell lung cancer (NSCLC) cell lines and patient tissues, suggesting that it may possess a carcinogenic role in lung cancer. This miRNA promoted the proliferation, migration and invasion of NSCLC cells by targeting and downregulating the expression of adenomatous polyposis coli (APC), which is a negative regulator of the canonical Wnt signaling pathway. Wnt signaling activation may increase transcription of miR20b. Therefore, miR20b and canonical Wnt signaling were coupled through a feedforward positive feedback loop, forming a biological regulatory circuit. Finally, an in vivo investigation further demonstrated that an increase in miR20b promoted the growth of cancer cells. Overall, our findings offer evidence that miR20b may contribute to the development of NSCLC by inhibiting APC via the canonical Wnt signaling pathway.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , Via de Sinalização Wnt/genética , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Retroalimentação Fisiológica , Feminino , Regulação Neoplásica da Expressão Gênica , Voluntários Saudáveis , Humanos , Pulmão/patologia , Pulmão/cirurgia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Pneumonectomia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Poliovirus receptor (PVR) is a tumor promoter and a regulatory checkpoint that enhances immunosuppression. We investigated PVR expression by applying immunohistochemistry (IHC) staining. A positive association existed between PVR expression and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) expression in patients with surgically resected non-small-cell lung cancer (NSCLC). PVR expression is a prognosis predictor of lung adenocarcinoma. PURPOSE: To investigate the prognostic significance of PVR expression and CTLA4 expression for surgically resected NSCLC. PATIENTS AND METHODS: The medical records of 108 Chinese patients with primary NSCLC who underwent surgery were retrospectively reviewed. The expression of PVR and CTLA4 were measured through IHC. Clinical characteristics, the association between PVR and CTLA4, and the prognostic significance of PVR were analyzed. RESULTS: A significant positive association was observed between PVR and CTLA4 expression in NSCLC (P = 0.016). PVR had a high positive rate among females, nonsmokers, and patients with adenocarcinoma and advanced lung cancer. The overall survival (OS) of patients with negative PVR expression was significantly longer than that of patients with positive PVR expression (P = 0.049), especially among females (P = 0.03) and nonsmokers (P = 0.025). Multivariate analysis results showed that advanced tumor stage and PVR expression were independent prognosis predictors of poor OS. CONCLUSION: PVR can potentially serve as a prognostic predictor and biomarker for NSCLC and cancer anti-CTLA4 immunotherapy response.
Assuntos
Antígeno CTLA-4/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores Virais/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
MicroRNAs regulate post-transcriptional gene expression and play important roles in multiple cellular processes. In this study, we found that miR-421 suppresses kelch-like ECH-associated protein 1(KEAP1) expression by targeting its 3'-untranslated region (3'UTR). A Q-PCR assay demonstrated that miR-421 is overexpressed in non-small cell lung cancer (NSCLC), especially in A549 cells. Consistently, the level of miR-421 was higher in clinical blood samples from lung cancer patients than in those from normal healthy donors, suggesting that miR-421 is an important lung cancer biomarker. Interestingly, overexpression of miR-421 reduced the level of KEAP1 expression, which further promoted lung cancer cell migration and invasion, as well as inhibited cell apoptosis both in vivo and in vitro. Furthermore, knockdown of miR-421 expression with an antisense morpholino oligonucleotide (AMO) increased ROS levels and treatment sensitivity to paclitaxel in vitro and in vivo, indicating that high miR-421 expression may at least partly account for paclitaxel tolerance in lung cancer patients. To find the upstream regulator of miR-421, one of the candidates, ß-catenin, was knocked out via the CRISPR/Cas9 method in A549 cells. Our data showed that inhibiting ß-catenin reduced miR-421 levels in A549 cells. In addition, ß-catenin upregulation enhanced miR-421 expression, indicating that ß-catenin regulates the expression of miR-421 in lung cancer. Taken together, our findings reveal the critical role of miR-421 in paclitaxel drug resistance and its upstream and downstream regulatory mechanisms. Therefore, miR-421 may serve as a potential molecular therapeutic target in lung cancer, and AMOs may be a potential treatment strategy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteína 1 Associada a ECH Semelhante a Kelch/genética , MicroRNAs/genética , Paclitaxel/administração & dosagem , Regiões 3' não Traduzidas/genética , Células A549 , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Xenoenxertos , Humanos , Masculino , Pessoa de Meia-Idade , Paclitaxel/efeitos adversosRESUMO
Non-small-cell lung cancer (NSCLC) is the predominant histological type of lung cancer and is characterized by the highest mortality and incidence rates among these types of malignancies. Cardiac glycosides, a class of natural products, have been identified as a potential type of chemotherapeutic agent. This study aims to investigate the anti-cancer effects and the mechanisms of action of Proscillaridin A (P.A) in NSCLC cells. In vitro sodium-potassium pump (Na+/K+ ATPase) enzyme assays indicated that P.A is a direct Na+/K+ ATPase inhibitor. P.A showed potent cytotoxic effects in NSCLC cells at nanomolar levels. Treatment mechanism studies indicated that P.A elevated Ca2+ levels, activated the AMPK pathway and downregulated phosphorylation of ACC and mTOR. Subsequently, P.A increased death receptor 4 (DR4) expression and downregulated NF-κB. Interestingly, P.A selectively suppressed EGFR activation in EGFR mutant cells but not in EGFR wild-type cells. In vivo, P.A significantly suppressed tumor growth in nude mice compared to vehicle-treated mice. Compared with the Afatinib treatment group, P.A displayed less pharmaceutical toxicity, as the body weight of mice treated with P.A did not decrease as much as those treated with Afatinib. Consistent changes in protein levels were obtained from western blotting analysis of tumors and cell lines. Immunohistochemistry analysis of the tumors from P.A-treated mice showed a significant suppression of EGFR phosphorylation (Tyr 1173) and reduction of the cell proliferation marker Ki-67. Taken together, our results suggest that P.A is a promising anti-cancer therapeutic candidate for NSCLC.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Proscilaridina/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Regulação para Cima/efeitos dos fármacos , Acetil-CoA Carboxilase/metabolismo , Adenilato Quinase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Pulmonares/metabolismo , Modelos Biológicos , Mutação/genética , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Proscilaridina/química , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant signaling transduction induced by mutant KRAS proteins occurs in 20â¼30% of non-small cell lung cancer (NSCLC), however, a direct and effective pharmacological inhibitor targeting KRAS has not yet reached the clinic to date. Honokiol, a small molecular polyphenol natural biophenolic compound derived from the bark of magnolia trees, exerts anticancer activity, however, its mechanism remains unknown. In this study, we sought to investigate the in vitro effects of honokiol on NSCLC cell lines harboring KRAS mutations. Honokiol was shown to induce G1 arrest and apoptosis to inhibit the growth of KRAS mutant lung cancer cells, which was weakened by an autophagy inhibitor 3-methyladenine (3-MA), suggesting a pro-apoptotic role of honokiol-induced autophagy that was dependent on AMPK-mTOR signaling pathway. In addition, we also discovered that Sirt3 was significantly up-regulated in honokiol treated KRAS mutant lung cancer cells, leading to destabilization of its target gene Hif-1α, which indicated that the anticancer property of honokiol maybe regulated via a novel mechanism associated with the Sirt3/Hif-1α. Taken together, these results broaden our understanding of the mechanisms on honokiol effects in lung cancer, and reinforce the possibility of its potential anticancer benefit as a popular Chinese herbal medicine (CHM).
RESUMO
Recently, microsomal prostaglandin E synthase 1 (mPGES-1) has attracted much attention from pharmacologists as a promising strategy and an attractive target for treating various types of diseases including rheumatoid arthritis (RA), which could preserve the anti-inflammatory effect while reducing the adverse effects often occur during administration of non-steroidal anti-inflammatory drugs (NSAIDs). Here, we report that sinomenine (SIN) decreased prostaglandin (PG)E2 levels without affecting prostacyclin (PG)I2 and thromboxane (TX)A2 synthesis via selective inhibiting mPGES-1 expression, a possible reason of low risk of cardiovascular event compared with NSAIDs. In addition, mPGES-1 protein expression was down-regulated by SIN treatment in the inflamed paw tissues both in carrageenan-induced edema model in rats and the collagen-II induced arthritis (CIA) model in DBA mice. More interestingly, SIN suppressed the last step of mPGES-1 gene expression by decreasing the DNA binding ability of NF-κB, paving a new way for drug discovery.