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OBJECTIVE: Oesophageal adenocarcinoma (EAC) arises in the setting of Barrett's oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic bile salts, the mimicry of reflux, in activation of NOTCH signaling in EAC. DESIGN: This study used public databases, EAC cell line models, L2-IL1ß transgenic mouse model and human EAC tissue samples to identify mechanisms of NOTCH activation under reflux conditions. RESULTS: Analysis of public databases demonstrated significant upregulation of NOTCH signaling components in EAC. In vitro studies demonstrated nuclear accumulation of active NOTCH1 cleaved fragment (NOTCH intracellular domain) and upregulation of NOTCH targets in EAC cells in response to reflux conditions. Additional investigations identified DLL1 as the predominant ligand contributing to NOTCH1 activation under reflux conditions. We discovered a novel crosstalk between APE1 redox function, reflux-induced inflammation and DLL1 upregulation where NF-κB can directly bind to and induce the expression of DLL1. The APE1 redox function was crucial for activation of the APE1-NF-κB-NOTCH axis and promoting cancer cell stem-like properties in response to reflux conditions. Overexpression of APE1 and DLL1 was detected in gastro-oesophageal junctions of the L2-IL1ß transgenic mouse model and human EAC tissue microarrays. DLL1 high levels were associated with poor overall survival in patients with EAC. CONCLUSION: These findings underscore a unique mechanism that links redox balance, inflammation and embryonic development (NOTCH) into a common pro-tumorigenic pathway that is intrinsic to EAC cells.
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Adenocarcinoma , Esôfago de Barrett , Neoplasias Esofágicas , Humanos , Camundongos , Animais , NF-kappa B/metabolismo , Neoplasias Esofágicas/patologia , Adenocarcinoma/patologia , Esôfago de Barrett/metabolismo , Camundongos Transgênicos , Oxirredução , InflamaçãoRESUMO
OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.
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Adenocarcinoma , Refluxo Gastroesofágico , Humanos , Animais , Camundongos , Metaloproteinase 14 da Matriz/metabolismo , Adenocarcinoma/patologia , Oxirredução , Transição Epitelial-Mesenquimal , Caderinas/metabolismo , Linhagem Celular TumoralRESUMO
PURPOSE: TGFß signaling is implicated in the progression of most cancers, including esophageal adenocarcinoma (EAC). Emerging evidence indicates that TGFß signaling is a key factor in the development of resistance toward cancer therapy. EXPERIMENTAL DESIGN: In this study, we developed patient-derived organoids and patient-derived xenograft models of EAC and performed bioinformatics analysis combined with functional genetics to investigate the role of SMAD family member 3 (SMAD3) in EAC resistance to oxaliplatin. RESULTS: Chemotherapy nonresponding patients showed enrichment of SMAD3 gene expression when compared with responders. In a randomized patient-derived xenograft experiment, SMAD3 inhibition in combination with oxaliplatin effectively diminished tumor burden by impeding DNA repair. SMAD3 interacted directly with protein phosphatase 2A (PP2A), a key regulator of the DNA damage repair protein ataxia telangiectasia mutated (ATM). SMAD3 inhibition diminished ATM phosphorylation by enhancing the binding of PP2A to ATM, causing excessive levels of DNA damage. CONCLUSIONS: Our results identify SMAD3 as a promising therapeutic target for future combination strategies for the treatment of patients with EAC.
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Adenocarcinoma , Reparo do DNA , Neoplasias Esofágicas , Oxaliplatina , Proteína Smad3 , Animais , Humanos , Camundongos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Organoides/efeitos dos fármacos , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The incidence of esophageal adenocarcinoma (EAC) has risen rapidly during the past four decades, making it the most common type of esophageal cancer in the USA and Western countries. The NEK (Never in mitosis A (NIMA) related kinase) gene family is a group of serine/threonine kinases with 11 members. Aberrant expression of NEKs has been recently found in a variety of human cancers and plays important roles in tumorigenesis, progression, and drug-resistance. However, the expression of the NEKs in EAC and its precancerous condition (Barrett's esophagus, BE) has not been investigated. In the present study, we first analyzed the TCGA and 9 GEO databases (a total of 10 databases in which 8 contain EAC and 6 contain BE) using bioinformatic approaches for NEKs expression in EAC and BE. We identified that several NEK members, such as NEK2 (7/8), NEK3 (6/8), and NEK6 (6/8), were significantly upregulated in EAC as compared to normal esophagus samples. Alternatively, NEK1 was downregulated in EAC as compared to the normal esophagus. On the contrary, genomic alterations of these NEKs are not frequent in EAC. We validated the above findings using qRT-PCR and the protein expression of NEKs in EAC cell lines using Western blotting and in primary EAC tissues using immunohistochemistry and immunofluorescence. Our data suggest that frequent upregulation of NEK2, NEK3, and NEK7 may be important in EAC.
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Esophageal adenocarcinoma (EAC), the predominant type of esophageal cancer in the United States, develops through Barrett's esophagus (BE)-dysplasia-carcinoma cascade. Gastroesophageal reflux disease, where acidic bile salts refluxate into the esophagus, is the main risk factor for the development of BE and its progression to EAC. The NFE2-related factor 2 (NRF2) is the master cellular antioxidant regulator. We detected high NRF2 protein levels in the EAC cell lines and primary tissues. Knockdown of NRF2 significantly enhanced acidic bile salt-induced oxidative stress, DNA damage, and inhibited EAC cell growth. Brusatol, an NRF2 inhibitor, significantly inhibited NRF2 transcriptional activity and downregulated the NRF2 target genes. We discovered that in addition to inducing apoptosis, Brusatol alone or in combination with cisplatin (CDDP) induced significant lipid peroxidation and ferroptosis, as evidenced by reduced xCT and GPX4 expression, two known ferroptosis markers. The combination of Brusatol and CDDP significantly inhibited EAC tumor xenograft growth in vivo and confirmed the in vitro data showing ferroptosis as an important mechanism in the tumors treated with Brusatol or Brusatol and CDDP combination. Our data support the role of NRF2 in protecting against stress-induced apoptosis and ferroptosis in EACs. Targeting NRF2 in combination with platinum therapy can be an effective strategy for eliminating cancer cells in EAC.
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Three-dimensional (3D) organoid culture systems are emerging as potential reliable tools to investigate basic developmental processes of human disease, especially cancer. The present study used established and modified culture conditions to report successful generation and characterization of patient-derived organoids from fresh primary tissue specimens of patients with treatment-naïve prostate cancer (PCa). Fresh tissue specimens were collected, digested enzymatically and the resulting cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Previously established 12-factor medium for organoid culturing was modified to create a minimal 5-factor medium. Organoids and corresponding tissue specimens were characterized using transcriptomic analysis, immunofluorescent analysis, and immunohistochemistry. Furthermore, patient-derived organoids were used to assess the drug response. Treatment-naïve patient-derived PCa organoids were obtained from fresh radical prostatectomy specimens. These PCa organoids mimicked the heterogeneity of corresponding parental tumor tissue. Histopathological analysis demonstrated similar tissue architecture and cellular morphology, as well as consistent immunohistochemical marker expression. Also, the results confirmed the potential of organoids as an in vitro model to assess potential personalized treatment responses as there was a differential drug response between different patient samples. In conclusion, the present study investigated patient-derived organoids from a cohort of treatment-naïve patients. Derived organoids mimicked the histological features and prostate lineage profiles of their corresponding parental tissue and may present a potential model to predict patient-specific treatment response in a pre-clinical setting.
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BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality. Cancer stem cells (CSCs) in CRC, which are spared by many chemotherapeutics, have tumorigenic capacity and are believed to be the reason behind cancer relapse. So far, there have been no effective drugs to target colon CSCs. Diiminoquinone (DIQ) has shown promising effects on targeting colon cancer. However, there is limited research on the effects of DIQ on eradicating CSCs in CRC. AIM: To investigate the anticancer potential of DIQ on colon CSCs in two-dimensional (2D) and three-dimensional (3D) models using colonospheres and patient-derived organoids. METHODS: Various 2D methods have been used to assess the effect and the mechanism of DIQ on HCT116 and HT29 cell lines including cell proliferation and viability assays, migration and invasion assays, immunofluorescence staining, and flow cytometry. The potency of DIQ was also assessed in 3D culture using the sphere formation assay and colon cancer patient-derived organoid model. RESULTS: Our results showed that DIQ significantly inhibited cell proliferation, migration, and invasion in HCT116 and HT29 cell lines. DIQ treatment induced apoptosis along with an accumulation of HCT116 and HT29 cancer cells in the sub-G1 region and an increase in reactive oxygen species in both CRC cell lines. DIQ reduced sphere-forming and self-renewal ability of colon cancer HCT116 and HT29 stem/progenitor cells at sub-toxic doses of 1 µmol/L. Mechanistically, DIQ targets CSCs by downregulating the main components of stem cell-related -catenin, AKT, and ERK oncogenic signaling pathways. Potently, DIQ displayed a highly significant decrease in both the count and the size of the organoids derived from colon cancer patients as compared to control and 5-fluorouracil conditions. CONCLUSION: This study is the first documentation of the molecular mechanism of the novel anticancer therapeutic DIQ via targeting CSC, a promising compound that needs further investigation.
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Neoplasias do Colo , Neoplasias Colorretais , Cateninas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Fluoruracila/farmacologia , Células HCT116 , Células HT29 , Humanos , Recidiva Local de Neoplasia , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Esophageal adenocarcinoma (EAC) is characterized by poor prognosis and low survival rate. Chronic gastroesophageal reflux disease (GERD) is the main risk factor for the development of Barrett's esophagus (BE), a preneoplastic metaplastic condition, and its progression to EAC. Yes-associated protein 1 (YAP1) activation mediates stem-like properties under cellular stress. The role of acidic bile salts (ABS) in promoting YAP1 activation under reflux conditions remains unexplored. METHODS: A combination of EAC cell lines, transgenic mice, and patient-derived xenografts were utilized in this study. mRNA expression and protein levels of APE1 and YAP1 were evaluated by qRT-PCR, western blot, and immunohistochemistry. YAP1 activation was confirmed by immunofluorescence staining and luciferase transcriptional activity reporter assay. The functional role and mechanism of regulation of YAP1 by APE1 was determined by sphere formation assay, siRNA mediated knockdown, redox-specific inhibition, and co-immunoprecipitation assays. RESULTS: We showed that YAP1 signaling is activated in BE and EAC cells following exposure to ABS, the mimicry of reflux conditions in patients with GERD. This induction was consistent with APE1 upregulation in response to ABS. YAP1 activation was confirmed by its nuclear accumulation with corresponding up-regulation of YAP1 target genes. APE1 silencing inhibited YAP1 protein induction and reduced its nuclear expression and transcriptional activity, following ABS treatment. Further investigation revealed that APE1-redox-specific inhibition (E3330) or APE1 redox-deficient mutant (C65A) abrogated ABS-mediated YAP1 activation, indicating an APE1 redox-dependent mechanism. APE1 silencing or E3330 treatment reduced YAP1 protein levels and diminished the number and size of EAC spheroids. Mechanistically, we demonstrated that APE1 regulated YAP1 stability through interaction with ß-TrCP ubiquitinase, whereas APE1-redox-specific inhibition induced YAP1 poly-ubiquitination promoting its degradation. CONCLUSION: Our findings established a novel function of APE1 in EAC progression elucidating druggable molecular vulnerabilities via targeting APE1 or YAP1 for the treatment of EAC.
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Adenocarcinoma , Esôfago de Barrett , Neoplasias Esofágicas , Refluxo Gastroesofágico , Adenocarcinoma/patologia , Animais , Esôfago de Barrett/genética , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Ácidos e Sais Biliares , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Neoplasias Esofágicas/patologia , Refluxo Gastroesofágico/complicações , Humanos , Camundongos , Oxirredução , Proteínas de Sinalização YAPRESUMO
Resistance of cancer cells and normal tissue toxicity of ionizing radiation (IR) are known to limit the success of radiotherapy. There is growing interest in using IR with natural compounds to sensitize cancer cells and spare healthy tissues. Thymoquinone (TQ) was shown to radiosensitize several cancers, yet no studies have investigated its radiosensitizing effects on colorectal cancer (CRC). Here, we combined TQ with IR and determined its effects in two-dimensional (2D) and three-dimensional (3D) culture models derived from HCT116 and HT29 CRC cells, and in patient-derived organoids (PDOs). TQ sensitized CRC cells to IR and reduced cell viability and clonogenic survival and was non-toxic to non-tumorigenic intestinal cells. TQ sensitizing effects were associated with G2/M arrest and DNA damage as well as changes in key signaling molecules involved in this process. Combining a low dose of TQ (3 µM) with IR (2 Gy) inhibited sphere formation by 100% at generation 5 and this was associated with inhibition of stemness and DNA repair. These doses also led to ~1.4- to ~3.4-fold decrease in organoid forming ability of PDOs. Our findings show that combining TQ and IR could be a promising therapeutic strategy for eradicating CRC cells.
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Unfolded protein response (UPR) protects malignant cells from endoplasmic reticulum stress-induced apoptosis. We report that Aurora kinase A (AURKA) promotes cancer cell survival by activating UPR in esophageal adenocarcinoma (EAC). A strong positive correlation between AURKA and binding immunoglobulin protein (BIP) mRNA expression levels was found in EACs. The in vitro assays indicated that AURKA promoted IRE1α protein phosphorylation, activating prosurvival UPR in FLO-1 and OE33 cells. The use of acidic bile salts to mimic reflux conditions in patients induced high AURKA and IRE1α levels. This induction was abrogated by AURKA knockdown in EAC cells. AURKA and p-IRE1α protein colocalization was observed in neoplastic gastroesophageal lesions of the L2-IL1b mouse model of Barrett's esophageal neoplasia. The combined treatment using AURKA inhibitor and tunicamycin synergistically induced cancer cell death. The use of alisertib for AURKA inhibition in the EAC xenograft model led to a decrease in IRE1α phosphorylation with a significant reduction in tumor growth. These results indicate that AURKA activates UPR, promoting cancer cell survival during ER stress in EAC. Targeting AURKA can significantly reverse prosurvival UPR signaling mechanisms and decrease cancer cell survival, providing a promising approach for the treatment of EAC patients.
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BACKGROUND: Glioblastoma multiforme (GBM), a stage IV astrocytoma, is the most common brain malignancy among adults. Conventional treatments of surgical resection followed by radio and/or chemotherapy fail to completely eradicate the tumor. Resistance to the currently available therapies is mainly attributed to a subpopulation of cancer stem cells (CSCs) present within the tumor bulk that self-renew leading to tumor relapse with time. Therefore, identification of characteristic markers specific to these cells is crucial for the development of targeted therapies. Glycogen synthase kinase 3 (GSK-3), a serine-threonine kinase, is deregulated in a wide range of diseases, including cancer. In GBM, GSK-3ß is overexpressed and its suppression in vitro has been shown to induce apoptosis of cancer cells. METHODS: In our study, we assessed the effect of GSK-3ß inhibition with Tideglusib (TDG), an irreversible non-ATP competitive inhibitor, using two human GBM cell lines, U-251 MG and U-118 MG. In addition, we combined TDG with radiotherapy to assess whether this inhibition enhances the effect of standard treatment. RESULTS: Our results showed that TDG significantly reduced cell proliferation, cell viability, and migration of both GBM cell lines in a dose- and time-dependent manner in vitro. Treatment with TDG alone and in combination with radiation significantly decreased the colony formation of U-251 MG cells and the sphere formation of both cell lines, by targeting and reducing their glioblastoma cancer stem-like cells (GSCs) population. Finally, cells treated with TDG showed an increased level of unrepaired radio-induced DNA damage and, thus, became sensitized toward radiation. CONCLUSIONS: In conclusion, TDG has proven its effectiveness in targeting the cancerous properties of GBM in vitro and may, hence, serve as a potential adjuvant radio-therapeutic agent to better target this deadly tumor.
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Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/terapia , Quimiorradioterapia Adjuvante/métodos , Glioblastoma/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Tiadiazóis/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Relação Dose-Resposta a Droga , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Ensaio Tumoral de Célula-TroncoRESUMO
BACKGROUND: Neuroblastoma (NB) is the most frequently diagnosed extracranial solid tumor among the pediatric population. It is an embryonic tumor with high relapse rates pertaining to the presence of dormant slowly dividing cancer stem cells (CSC) within the tumor bulk that are responsible for therapy resistance. Therefore, there is a dire need to develop new therapeutic approaches that specifically target NB CSCs. Glycogen synthase kinase (GSK)-3ß is a serine/threonine kinase that represents a common signaling node at the intersection of many pathways implicated in NB CSCs. GSK-3ß sustains the survival and maintenance of CSCs and renders them insensitive to chemotherapeutic agents and radiation. METHODS: In our study, we aimed at evaluating the potential anti-tumor effect of Tideglusib (TDG), an irreversible GSK-3ß inhibitor drug, on three human NB cell lines, SK-N-SH, SH-SY5Y, and IMR-32. RESULTS: Our results showed that TDG significantly reduced cell proliferation, viability, and migration of the NB cells, in a dose- and time-dependent manner, and also significantly hindered the neurospheres formation eradicating the self-renewal ability of highly resistant CSCs. Besides, TDG potently reduced CD133 cancer stem cell marker expression in both SH-SY5Y cells and G1 spheres. Lastly, TDG inhibited NB tumor growth and progression in vivo. CONCLUSION: Collectively, we concluded that TDG could serve as an effective treatment capable of targeting the NB CSCs and hence overcoming therapy resistance. Yet, future studies are warranted to further investigate its potential role in NB and decipher the subcellular and molecular mechanisms underlying this role.
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Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Tiadiazóis/uso terapêutico , Antígeno AC133/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Humanos , Camundongos , Cicatrização/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Coronavirus disease 2019 (COVID-19) has infected millions of people worldwide and emerged to be the biggest global health threat claiming hundreds of thousands of lives at exponential rates. The severity of the disease increases with old age and presence of underlying health conditions, such as cancer. Managing cancer patients under these circumstances is rather challenging, given their compromised immunity and the overwhelmed health care services by COVID-19 community transmission. Thus, it is prudent to establish common guidelines for the monitoring and treatment of cancer patients. In this review, we comprehensively investigate the various aspects of cancer care during the COVID-19 pandemic, discuss challenges faced while treating cancer patients, and propose potential approaches to manage COVID-19 among this vulnerable population. We also discuss molecular aberrations and genetic changes associated with cancer and their role in affecting the virus' infectivity and severity. Lastly, we shed light on therapeutic approaches that can encompass both diseases without compromising one over the other.
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BACKGROUND: Prostate cancer (PC) is the most frequently diagnosed cancer among men worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We interrogated proteomic signatures that embody the transition of PC from an androgen-dependent (AD) to an androgen-independent (AI) state. METHODS: We have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC at phenotypic and subcellular levels. We statistically surveyed global protein expression profiles in these cell lines. Differential profiles were functionally interrogated by pathways and protein-protein interaction network analyses. RESULTS: Protein expression pattern analysis revealed a total of 683 proteins, among which 99 were significantly differentially altered in PLum-AI cells as compared to PLum-AD cells (45 increased and 54 decreased). Principal component analysis (PCA) revealed that the two different cell lines clearly separated apart, indicating a significant proteome expression difference between them. Four of the proteins (vimentin, catalase, EpCAM, and caspase 3) that were differentially expressed in PLum-AI cells compared to PLum-AD cells were subjected to biochemical validation by Western blotting. Biological process gene ontology (GO) analysis of the differentially expressed proteins demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to PI3 kinase and androgen receptor pathways. Besides, other relevant biological processes that are enriched in PLum-AI cells included cell adhesion and cell migration processes, cell and DNA damage, apoptosis, and cell cycle regulation. CONCLUSIONS: Our protein expression analysis of a murine in vitro model of PC progression identified differential protein spots that denote this progression and that comprise high-potential targets for early treatment of PC with a personalized patient-specific approach. Efforts are underway to functionally assess the potential roles of these proteins as therapeutic targets for PC progression.
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BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among adult males globally. The poor prognosis of PCa is largely due to late diagnosis of the disease when it has already progressed to an advanced stage marked by androgen-independence, thus necessitating new strategies for early detection and treatment. We construe that these direly needed advances are limited by our poor understanding of early events in the progression of PCa and that would thus represent ideal targets for early intervention. To begin to fill this void, we interrogated molecular "oncophenotypes" that embody the transition of PCa from an androgen-dependent (AD) to-independent (AI) state. METHODS: To accomplish this aim, we used our previously established AD and AI murine PCa cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PCa morphologically and molecularly. We statistically surveyed global gene expressions in these cell lines by microarray analysis. Differential profiles were functionally interrogated by pathways, gene set enrichment and topological gene network analyses. RESULTS: Gene expression analysis of PLum-AD and PLum-AI transcriptomes (n = 3 each), revealed 723 differentially expressed genes (392 upregulated and 331 downregulated) in PLum-AI compared to PLum-AD cells. Gene set analysis demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to tumor aggressiveness including cell migration and invasion facilitated by epithelial-to-mesenchymal transition (EMT). Further analysis demonstrated that the p38 mitogen-activated protein kinase (MAPK) was predicted to be significantly activated in the PLum-AI cells, whereas gene sets previously associated with favorable response to the p38 inhibitor SB203580 were attenuated (i.e., inversely enriched) in the PLum-AI cells, suggesting that these aggressive cells may be therapeutically vulnerable to p38 inhibition. Gene set and gene-network analysis also alluded to activation of other signaling networks particularly those associated with enhanced EMT, inflammation and immune function/response including, but not limited to Tnf, IL-6, Mmp 2, Ctgf, and Ptges. Accordingly, we chose SB203580 and IL-6 to validate their effect on PLum-AD and PLum-AI. Some of the common genes identified in the gene-network analysis were validated at the molecular and functional level. Additionally, the vulnerability to SB203580 and the effect of IL-6 were also validated on the stem/progenitor cell population using the sphere formation assay. CONCLUSIONS: In summary, our study highlights pathways associated with an augmented malignant phenotype in AI cells and presents new high-potential targets to constrain the aggressive malignancy seen in the castration-resistant PCa.
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Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Interleucina-6/farmacologia , Neoplasias da Próstata/patologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Androgênios/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Imidazóis/uso terapêutico , Interleucina-6/uso terapêutico , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Piridinas/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The high recurrence rates of colorectal cancer have been associated with a small population of cancer stem cells (CSCs) that are resistant to the standard chemotherapeutic drug, 5-fluorouracil (5FU). Thymoquinone (TQ) has shown promising antitumor properties on numerous cancer systems both in vitro and in vivo; however, its effect on colorectal CSCs is poorly established. Here, we investigated TQ's potential to target CSCs in a three-dimensional (3D) sphere-formation assay enriched for a population of colorectal cancer stem/progenitor cells. Our results showed a significant decrease in self-renewal potential of CSC populations enriched from 5FU-sensitive and resistant HCT116 cells at 10-fold lower concentrations when compared to 2D monolayers. TQ decreased the expression levels of colorectal stem cell markers CD44 and Epithelial Cell Adhesion Molecule EpCAM and proliferation marker Ki67 in colonospheres derived from both cell lines and reduced cellular migration and invasion. Further investigation revealed that TQ treatment led to increased TUNEL positivity and a dramatic increase in the amount of the DNA damage marker gamma H2AX particularly in 5FU-resistant colonospheres, suggesting that the diminished sphere forming ability in TQ-treated colonospheres is due to induction of DNA damage and apoptotic cell death. The intraperitoneal injection of TQ in mice inhibited tumor growth of spheres derived from 5FU-sensitive and 5FU-resistant HCT116 cells. Furthermore, TQ induced apoptosis and inhibited NF-κB and MEK signaling in mouse tumors. Altogether, our findings document TQ's effect on colorectal cancer stem-like cells and provide insights into its underlying mechanism of action.
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Chemotherapy has been shown to be effective in reducing the progression and development of cancer in metastatic patients. However, drug selectivity is still a major issue for most chemotherapeutics. In this study, we synthesized four novel heterocyclic compounds having similarity in structure with quinone systems whereby nitrogen atoms replace the oxygen atoms. The anticancer activity of these compounds (DIQ3-6) was tested against HCT116 human colon cancer cells. We showed that all four heterocycles caused significant reduction in colon cancer cell viability at doses as low as 4 µM, a concentration that was not cytotoxic to normal human FHs74Int intestinal cell lines. Interestingly, these heterocycles inhibited colon sphere formation in 3D cultures at first generation (G1), mainly because of inhibition of proliferation as evidenced by Ki67 staining. Thus, DIQ3 causes sufficient eradication of the self-renewal ability of the highly resistant cancer stem cells. This study represents the first documentation of the activity of these novel heterocyclic compounds, particularly compound DIQ3, and their potential therapeutic use in targeting colon cancer self-renewal capacity. Our findings provide the basis for proposing these nontoxic and stable compounds for additional testing against cancer.
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Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here, we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both, Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells, but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly, two principle stem cell regulatory pathways WNT/ß-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore, both treatments strikingly eliminated CD133+ CSC population, accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear ß-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of ß-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU, both, Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together, combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells.
Assuntos
Benzoquinonas/farmacologia , Neoplasias Colorretais/genética , Citotoxinas/farmacologia , Fluoruracila/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Antígeno AC133/metabolismo , Animais , Benzoquinonas/química , Adesão Celular/efeitos dos fármacos , Embrião de Galinha , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citotoxinas/química , Fluoruracila/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Organoides/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
Thymoquinone (TQ), the active ingredient of black seed, is a promising anticancer molecule that inhibits cancer cell growth and progression in vitro and in vivo. Despite the promising anticancer activities of TQ, its translation to the clinic is limited by its poor bioavailability and hydrophobicity. As such, we and others encapsulated TQ in nanoparticles to improve its delivery and limit undesirable cytotoxicity. These TQ-nanoparticle formulations showed improved anticancer and anti-inflammatory activities when compared with free TQ. Here, we provide an overview of the various TQ-nanoparticle formulations, highlight their superior efficacy and discuss up-to-date solutions to further enhance TQ bioavailability and anticancer activity, thus improving potential for clinical translation.
Assuntos
Antineoplásicos/administração & dosagem , Benzoquinonas/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Benzoquinonas/uso terapêutico , Humanos , Nanopartículas/uso terapêutico , NanotecnologiaRESUMO
Cancer Stem Cells (CSCs) are a sub-population of cells, identified in most tumors, responsible for the initiation, recurrence, metastatic potential, and resistance of different malignancies. In prostate cancer (PCa), CSCs were identified and thought to be responsible for the generation of the lethal subtype, commonly known as Castration-Resistant Prostate Cancer (CRPC). In vitro models to investigate the properties of CSCs in PCa are highly required. Sphere-formation assay is an in vitro method commonly used to identify CSCs and study their properties. Here, we report the detailed methodology on how to generate and propagate spheres from PCa cell lines and from murine prostate tissue. This model is based on the ability of stem cells to grow in non-adherent serum-free gel matrix. We also describe how to use these spheres in histological and immuno-fluorescent staining assays to assess the differentiation potential of the CSCs. Our results show the sphere-formation Assay (SFA) as a reliable in vitro assay to assess the presence and self-renewal ability of CSCs in different PCa models. This platform presents a useful tool to evaluate the effect of conventional or novel agents on the initiation and self-renewing properties of different tumors. The effects can be directly evaluated through assessment of the sphere-forming efficiency (SFE) over five generations or other downstream assays such as immuno-histochemical analysis of the generated spheres.