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Inflammation and obesity are two major factors that promote Colorectal cancer (CRC). Our recent data suggests that interleukin (IL)-23, is significantly elevated in CRC tumors and correlates with patient obesity, tumor grade and survival. Thus, we hypothesize that obesity and CRC may be linked via inflammation and IL-23 may be a potential target for intervention in high-risk patients. TCGA dataset and patient sera were evaluated for IL-23A levels. IL-23A [IL-23 p19-/-] knockout (KO) mice were crossed to Apcmin/+ mice and progeny were fed low-fat or high-fat diets. At termination intestines were evaluated for tumorigenesis. Tumors, serum, and fecal contents were analyzed for protein biomarkers, cytokines, and microbiome profile respectively. IL-23A levels are elevated in the sera of patients with obesity and colon tumors. Genetic ablation of IL-23A significantly suppressed colonic tumor multiplicity (76-96 %) and incidence (72-95 %) in male and female mice. Similarly, small-intestinal tumor multiplicity and size were also significantly reduced in IL-23A KO mice. IL-23A knockdown in Apcmin/+ mice fed high-fat diet, also resulted in significant suppression of colonic (50-58 %) and SI (41-48 %) tumor multiplicity. Cytokine profiling showed reduction in several circulating pro-inflammatory cytokines including loss of IL-23A. Biomarker analysis suggested reduced tumor cell proliferation and immune modulation with an increase in tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the IL-23A KO mice compared to controls. Fecal microbiome analysis revealed potentially beneficial changes in the bacterial population profile. In summary, our data indicates a tumor promoting role for IL-23 in CRC including diet-induced obesity. With several IL-23 targeted therapies in clinical trials, there is a great potential for targeting this cytokine for CRC prevention and therapy.
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Neoplasias do Colo , Neoplasias Colorretais , Animais , Feminino , Humanos , Masculino , Camundongos , Neoplasias do Colo/patologia , Neoplasias Colorretais/genética , Citocinas , Inflamação , Interleucina-23/genética , Interleucina-23/efeitos adversos , Subunidade p19 da Interleucina-23 , Camundongos Knockout , Obesidade/genéticaRESUMO
Colorectal cancer (CRC) incidence is rising globally. Hence, preventing this disease is a high priority. With this aim, we determined the CRC prevention potential of the TRAIL-inducing small molecule ONC201/TIC10 using a preclinical model representing high-risk familial adenomatous polyposis (FAP) patients, Apc min/+ mice. Prior to the efficacy study, optimal and non-toxic doses of ONC201 were determined by testing five different doses of ONC201 (0-100 mg/kg body weight (BW); twice weekly by oral gavage) in C57BL/6J mice (n=6/group) for 6 weeks. BW gain, organ weights and histopathology, blood profiling, and the plasma liver enzyme profile suggested no toxicities of ONC201 at doses up to 100 mg/kg BW. For efficacy determination, beginning at six weeks of age, groups of Apc min/+ male and female mice (n≥20) treated with colon carcinogen azoxymethane (AOM) (AOM-Apc min/+) were administered ONC201 (0, 25, and 50 mg/kg BW) as above up to 20 weeks of age. At termination, efficacy was determined by comparing the incidence and multiplicity of intestinal tumors between vehicle- and drug-treated groups. ONC201 showed a strong suppressive effect against the development of both large and small intestinal tumors in male and female mice. Apc min/+ mice treated with ONC201 (50 mg/kg BW) showed >50% less colonic tumor incidence (P<0.0002) than controls. Colonic tumor multiplicity was also significantly reduced by 68% in male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in controls; P<0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in controls; P<0.0003) with ONC201 treatment (50 mg/kg BW). Small intestinal polyps were reduced by 68% in male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in controls; P<0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in controls; P<0.0001). Molecular analysis of the tumors suggested an increase in TRAIL, DR5, cleaved caspases 3/7/8, Fas-associated death domain protein (FADD), and p21 (WAF1) in response to drug treatment. Serum analysis indicated a decrease in pro-inflammatory serum biomarkers, such as IL1ß, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated mice compared with controls. Our data demonstrated excellent chemopreventive potential of orally administered ONC201 against intestinal tumorigenesis in the AOM-Apc min/+ mouse model.
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Obesity-associated chronic inflammation predisposes colon cancer risk development. Interleukin-23 (IL-23) is a potential inflammatory mediator linking obesity to chronic colonic inflammation, altered gut microbiome, and colon carcinogenesis. We aimed to elucidate the role of pro-inflammatory eicosanoids and gut bacterial toxins in priming dendritic cells and macrophages for IL-23 secretion to promote colon tumor progression. To investigate the association of IL-23 with obesity and colon tumorigenesis, we utilized TCGA data set and colonic tumors from humans and preclinical models. To understand IL-23 production by inflammatory mediators and gut microbial toxins, we performed several in vitro mechanistic studies to mimic the tumor microenvironment. Colonic tumors were utilized to perform the ex vivo experiments. Our findings showed that IL-23 is elevated in obese individuals, colonic tumors and correlated with reduced disease-free survival. In vitro studies showed that IL-23 treatment increased the colon tumor cell self-renewal, migration, and invasion while disrupting epithelial barrier permeability. Co-culture experiments of educated dendritic cells/macrophages with colon cancer cells significantly increased the tumor aggression by increasing the secretory levels of IL-23, and these observations are further supported by ex vivo rat colonic tumor organotypic experiments. Our results demonstrate gut microbe toxins and eicosanoids facilitate IL-23 production, which plays an important role in obesity-associated colonic tumor progression. This newly identified nexus represents a potential target for the prevention and treatment of obesity-associated colon cancer.
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Colorectal cancer causes over 53,000 deaths annually in the United States. Its rising incidences worldwide and particularly in young adults is a major concern. Here, we evaluated the efficacy of omeprazole that is clinically approved for treating acid reflux, to enable its repurposing for colorectal cancer prevention. In the azoxymethane-induced rat colorectal cancer model, dietary omeprazole (250 and 500 ppm) was administered at early adenoma stage (8 weeks after azoxymethane) to assess the progression of early lesions to adenocarcinoma. Administration of omeprazole at 250 or 500 ppm doses led to suppression of total colon adenocarcinoma incidence by 15.7% and 32% (P < 0.01), respectively. Importantly, invasive carcinoma incidence was reduced by 59% (P < 0.0005) and 90% (P < 0.0001) in omeprazole-administered rats in a dose-dependent manner. There was also a strong and dose-dependent inhibition in the adenocarcinoma multiplicity in rats exposed to omeprazole. Administration of 250 and 500 ppm omeprazole inhibited total colon adenocarcinoma multiplicity by approximately 49% and approximately 65% (P < 0.0001), respectively. While noninvasive adenocarcinomas multiplicity was suppressed by approximately 34% to approximately 48% (P < 0.02), the invasive carcinomas multiplicity was reduced by approximately 74% to approximately 94% (P < 0.0001) in omeprazole-exposed rats in comparison with the untreated rats. Biomarker analysis results showed a decrease in cell proliferation and anti-apoptotic/pro-survival proteins with an increase in apoptosis. Transcriptome analysis of treated tumors revealed a significant increase in adenocarcinoma inhibitory genes (Olmf4; Spink4) expression and downregulation of progression promoting genes (SerpinA1, MMP21, IL6). In summary, omeprazole showed significant protection against the progression of adenoma to adenocarcinoma. PREVENTION RELEVANCE: Preventing colon cancer is urgently needed because of its high incidence and mortality rates worldwide. Toward this end, preventive efficacy of omeprazole, a common medication, was evaluated in animal model of colorectal cancer and was found to suppress colonic adenoma progression to carcinoma. These findings warrant its further evaluation in humans.
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Adenocarcinoma , Adenoma , Neoplasias do Colo , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/metabolismo , Adenocarcinoma/prevenção & controle , Adenoma/induzido quimicamente , Adenoma/prevenção & controle , Animais , Azoximetano/toxicidade , Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/prevenção & controle , Omeprazol/efeitos adversos , Inibidores da Bomba de Prótons/efeitos adversos , Ratos , Ratos Endogâmicos F344RESUMO
PURPOSE: Triple-negative breast cancer (TNBC) is the most challenging and aggressive subtype of breast cancer with limited treatment options because of tumor heterogeneity, lack of druggable targets and therapy resistance. TNBCs are characterized by overexpression of growth factor receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) making them promising therapeutic targets. Regorafenib is an FDA approved oral multi-kinase inhibitor that blocks the activity of multiple protein kinases including those involved in the regulation of tumor angiogenesis [VEGFR1-3, TIE2], tumor microenvironment [PDGFR-ß, FGFR] and oncogenesis (KIT, RET, RAF-1, BRAF). In the current study, we examined the radiosensitizing effects of Regorafenib on TNBC cell lines and explored the mechanism by which Regorafenib enhances radiosensitivity. METHODS: MDA-MB-231 and SUM159PT (human TNBC cell lines) and MCF 10a (human mammary epithelial cell line) were treated with Regorafenib, ionizing radiation or a combination of both. Following treatment with Regorafenib and radiation we conducted clonogenic assay to determine radiosensitivity, immunoblot analysis to assess the effect on key signaling targets, tube formation to evaluate effect on angiogenesis and comet assay as well as western blot for γH2AX to assess DNA damage response (DDR). RESULTS: Regorafenib reduced cell proliferation and enhanced radiosensitivity of MDA-MB-231 and SUM159PT cell lines but had no effect on the MCF 10a cells. Clonogenic survival assays showed that the surviving fraction at 2 Gy for both MDA-MB-231 and SUM159PT was reduced from 66.4 ± 8.9 and 88.2 ± 1.7 in controls to 38.1 ± 4.9 and 75.1 ± 1.1 following a 24 hr pretreatment with 10 µM and 5 µM Regorafenib, respectively. A marked reduction in the expression of VEGFR, PDGFR, EGFR and the downstream target, ERK, was observed with Regorafenib treatment alone or in combination with radiation. We also observed a significant inhibition of VEGF-A production in the TNBC cell lines following treatment with Regorafenib. Further, the addition of conditioned medium from Regorafenib-treated tumor cells onto human umbilical vein endothelial cells (HUVEC) suppressed tube formation, indicating an inhibition of tumor angiogenesis. Regorafenib also decreased migration of TNBC cells and suppressed radiation-induced DNA damage repair in a time-dependent manner. CONCLUSIONS: Our findings demonstrate that Regorafenib enhanced radiosensitivity of breast cancer cells by inhibiting the expression of multiple receptor tyrosine kinases, VEGF-mediated angiogenesis and DNA damage response in TNBC. Therefore, combining Regorafenib with radiation and antiangiogenic agents will be beneficial and effective in controlling TNBC.
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Dano ao DNA , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , HumanosRESUMO
Chemoresistance cells have features similar to cancer stem cells. Elimination of these cells is an effective therapeutic strategy to clinically combat chemoresistance non-small cell lung cancer (NSCLC). Here, we demonstrate that Doublecortin-like kinase1 (DCLK1) is the key to developing chemoresistance and associated stemness in NSCLC. DCLK1 is highly expressed in human lung adenocarcinoma and strongly correlated with stemness. Silencing DCLK1 inhibits NSCLC cell primary and secondary spheroid formation, which is the prerequisite feature of tumor stem cells. DCLK1 inhibition reduced NSCLC cell migration/invasion in vitro and induced tumor growth inhibition in vivo. NSCLC cells responded differently to cisplatin treatment; indeed, the clonogenic ability of all NSCLC cells was reduced. We found that the cisplatin-resistant NSCLC cells gain the expression of DCLK1 compared with their parental control. However, DCLK1 inhibition in cisplatin-resistance NSCLC cells reverses the tumor cell resistance to cisplatin and reduced tumor self-renewal ability. Specifically, we found that DCLK1-mediated cisplatin resistance in NSCLC is via an ABC subfamily member 4 (ABCD4)-dependent mechanism. Our data demonstrate that increased expression of DCLK1 is associated with chemoresistance and enhanced cancer stem cell-like features in NSCLC. Targeting DCLK1 using gene knockdown/knockout strategies alone or in combination with cisplatin may represent a novel therapeutic strategy to treat NSCLC.
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Tumor-associated M2-macrophages are one of the most abundant immunosuppressive cell types in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the molecular mechanisms responsible for the generation of M2-macrophages are unclear. Here, we demonstrated that overexpression of DCLK1-isoform2 in AsPC1 and MIA PaCa2 cells resulted in the polarization of M1-macrophages toward an M2 phenotype via secreted chemokines/cytokines. These M2-macrophages enhanced parental PDAC cell migration, invasion, and self-renewal, and this was associated with increased expression of Snail and Slug. We observed distinct expression of Dclk-isoform2, marked infiltration of M2-macrophages, and a marginal increase of CD8+ T cells in 20-week-old KPCY mice pancreas compared with 5 weeks old. Utilizing an autochthonous mouse model of pancreatic adenocarcinoma, we observed distinct immunoreactive Dclk1 and arginase1 in tissues where CD8+ T-cell infiltration was low and observed a paucity of DCLK1 and arginase1 staining where CD8+ T-cell infiltration was high. Finally, we found that DCLK1-isoform2 tumor-educated M2-macrophages inhibit CD8+ T-cell proliferation and granzyme-B activation. Inhibition of DCLK1 in an organoid coculture system enhanced CD8+ T-cell activation and associated organoid death. We conclude that DCLK1-isoform2 is a novel initiator of alternate macrophage activation that contributes to the immunosuppression observed in the PDAC TME. These data suggest that tumor DCLK1-isoform2 may be an attractive target for PDAC therapy, either alone or in conjunction with immunotherapeutic strategies.
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Processamento Alternativo , Carcinoma Ductal Pancreático/imunologia , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pancreáticas/imunologia , Proteínas Serina-Treonina Quinases/genética , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/prevenção & controle , Movimento Celular , Proliferação de Células , Quinases Semelhantes a Duplacortina , Humanos , Ativação de Macrófagos , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Isoformas de Proteínas , Microambiente TumoralRESUMO
BACKGROUND: Non-steroidal anti-inflammatory drugs, cyclooxygenase (COX)-2 selective inhibitors, have been explored for prevention and treatment of several inflammatory chronic conditions including arthritis, and cancer. However, the long-term use of these drugs is associated with gastrointestinal, renal, and cardiovascular side effects. Later, COX/5-lipoxygenase (5-LOX) dual inhibitors (eg, licofelone) have been developed but did not enter into the market from the clinical trails due to COX-1/2 inhibition-associated side effects. Hence, targeting microsomal prostaglandin E synthase-1 (mPGES-1) and 5-LOX can be an ideal approach while sparing COX-1/2 activities for development of the next generation of anti-inflammatory drugs with better efficacy and safety. MATERIALS AND METHODS: In silico (molecular modelling) studies were used to design a mPGES-1/5-LOX dual inhibitory and COX-1/2 sparing lead molecule licofelone analogue-9 (LFA-9) by modifying the pharmacophore of licofelone. In vitro cell-free enzymatic (mPGES-1, 5-LOX, COX-1/2) assays using fluorometric/colorimetric methods and cell-based assays (LPS-induced PGE2, LTB4, and PGI2 productions from macrophages) using ELISA technique, isothermal calorimetry, and circular dichroism techniques were performed to determine the mPGES-1/5-LOX inhibitory efficacy and selectivity. Anti-inflammatory efficacy of LFA-9 was evaluated using a carrageenan (inflammogen)-induced rat paw edema model. Infiltration/expression of CD68 immune cells and TNF-α in paw tissues were evaluated using confocal microscope and immunoblot analysis. Anti-cancer effect of LFA-9 was evaluated using colon spheroids in vitro. RESULTS: LFA-9 inhibited mPGES-1/5-LOX and their products PGE2 and LTB4, spared COX-1/2 and its product PGI2. LFA-9 bound strongly with human mPGES-1/5-LOX enzymes and induced changes in their secondary structure, thereby inhibited their enzymatic activities. LFA-9 inhibited carrageenan-induced inflammation (70.4%) in rats and suppressed CD68 immune cell infiltration (P ≤ 0.0001) and TNF-α expression. LFA-9 suppressed colon tumor stemness (60.2%) in vitro through inhibition of PGE2 (82%) levels. CONCLUSION: Overall study results suggest that LFA-9 is a mPGES-1/5-LOX dual inhibitor and showed anti-inflammatory and colorectal cancer preventive activities, and warranted detailed studies.
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Aberrant expression of GLI1 is responsible for aggressive tumor behavior and survival due to its effects on the DNA damage response (DDR). We investigated whether interleukin (IL)-24, a tumor suppressor, inhibits GLI1 and the associated DDR pathway in human NSCLCs. IL-24 treatment reduces mRNA and protein expression of GLI1 in lung tumor cells, but not in normal cells. GLI1 reporter assay and mRNA studies demonstrated that IL-24 regulates GLI1 at the post-transcriptional level by favoring mRNA degradation. Associated with GLI1 inhibition was marked suppression of the ATM-mediated DDR pathway resulting in increased DNA damage, as evidenced by γ-H2AX foci and Comet assay. Furthermore, attenuation of GLI1-associated DDR by IL-24 increased caspase-3 and PARP activity, resulting in cancer cell apoptosis. GLI1 inhibition and overexpression confirmed that IL-24-mediated anti-tumor effects involved the GLI-dependent pathway. Finally, we observed that IL-24-mediated alteration in GLI1 is independent of the canonical hedgehog-signaling pathway. Our study provides evidence that IL-24 treatment induces DNA damage, and reduces GLI1 expression and offers an opportunity for testing IL-24-based therapy for inhibiting GLI1 in lung cancer.
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Effective and safe delivery of anticancer agents is among the major challenges in cancer therapy. The majority of anticancer agents are toxic to normal cells, have poor bioavailability, and lack in vivo stability. Recent advancements in nanotechnology provide safe and efficient drug delivery systems for successful delivery of anticancer agents via nanoparticles. The physicochemical and functional properties of the nanoparticle vary for each of these anticancer agents, including chemotherapeutics, nucleic acid-based therapeutics, small molecule inhibitors, and photodynamic agents. The characteristics of the anticancer agents influence the design and development of nanoparticle carriers. This review focuses on strategies of nanoparticle-based drug delivery for various anticancer agents. Recent advancements in the field are also highlighted, with suitable examples from our own research efforts and from the literature.
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Antineoplásicos/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Humanos , Nanopartículas/químicaRESUMO
Co-administration of functionally distinct anti-cancer agents has emerged as an efficient strategy in lung cancer treatment. However, a specially designed drug delivery system is required to co-encapsulate functionally different agents, such as a combination of siRNA and chemotherapy, for targeted delivery. We developed a folic acid (FA)-conjugated polyamidoamine dendrimer (Den)-based nanoparticle (NP) system for co-delivery of siRNA against HuR mRNA (HuR siRNA) and cis-diamine platinum (CDDP) to folate receptor-α (FRA) -overexpressing H1299 lung cancer cells. The co-delivery of HuR siRNA and CDDP using the FRA-targeted NP had a significantly greater therapeutic effect than did individual therapeutics. Further, the FRA-targeted NP exhibited improved cytotoxicity compared to non-targeted NP against lung cancer cells. Finally, the NP showed negligible toxicity towards normal MRC9 lung fibroblast cells. Thus, the present study demonstrates FRA-targeted Den nanoparticle system as a suitable carrier for targeted co-delivery of siRNA and chemotherapy agents in lung cancer cells.
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Carcinoma Pulmonar de Células não Pequenas/terapia , Cisplatino/farmacologia , Dendrímeros/química , Sistemas de Liberação de Medicamentos , Proteína Semelhante a ELAV 1/antagonistas & inibidores , Receptor 1 de Folato/metabolismo , Nanopartículas/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Proteína Semelhante a ELAV 1/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Nanopartículas/química , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
The Hippo pathway is an evolutionarily conserved signaling pathway that regulates proliferation and apoptosis to control organ size during developmental growth. Yes-associated protein 1 (YAP1), the terminal effector of the Hippo pathway, is a transcriptional co-activator and a potent growth promoter that has emerged as a critical oncogene. Overexpression of YAP1 has been implicated in promoting resistance to chemo-, radiation and targeted therapy in various cancers. However, the role of YAP1 in radioresistance in triple-negative breast cancer (TNBC) is currently unknown. We evaluated the role of YAP1 in radioresistance in TNBC in vitro, using two approaches to inhibit YAP1: 1) genetic inhibition by YAP1 specific shRNA or siRNA, and 2) pharmacological inhibition by using the small molecule inhibitor, verteporfin that prevents YAP1 transcriptional activity. Our findings demonstrate that both genetic and pharmacological inhibition of YAP1 sensitizes TNBC cells to radiation by inhibiting the EGFR/PI3K/AKT signaling axis and causing an increased accumulation of DNA damage. Our results reveal that YAP1 activation exerts a protective role for TNBC cells in radiotherapy and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of TNBC.
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Successful chemotherapeutic intervention for management of lung cancer requires an efficient drug delivery system. Gold nanoparticles (GNPs) can incorporate various therapeutics; however, GNPs have limitations as drug carriers. Nano-sized cellular vesicles like exosomes (Exo) can ferry GNP-therapeutic complexes without causing any particle aggregation or immune response. In the present study, we describe the development and testing of a novel Exo-GNP-based therapeutic delivery system -'nanosomes'- for lung cancer therapy. This system consists of GNPs conjugated to anticancer drug doxorubicin (Dox) by a pH-cleavable bond that is physically loaded onto the exosomes (Exo-GNP-Dox). The therapeutic efficacy of Dox in nanosomes was assessed in H1299 and A549 non-small cell lung cancer cells, normal MRC9 lung fibroblasts, and Dox-sensitive human coronary artery smooth muscle cells (HCASM). The enhanced rate of drug release under acidic conditions, successful uptake of the nanosomes by the recipient cells and the cell viability assays demonstrated that nanosomes exhibit preferential cytotoxicity towards cancer cells and have minimal activity on non-cancerous cells. Finally, the underlying mechanism of cytotoxicity involved ROS-mediated DNA damage. Results from this study mark the establishment of an amenable drug delivery vehicle and highlight the advantages of a natural drug carrier that demonstrates reduced cellular toxicity and efficient delivery of therapeutics to cancer cells.
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Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Exossomos/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas Metálicas/química , Antineoplásicos/farmacologia , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Vasos Coronários/patologia , Dano ao DNA , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Cinética , Neoplasias Pulmonares/patologia , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/ultraestrutura , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Interleukin (IL)-24, a novel tumor suppressor/cytokine exhibits antitumor activity against a broad-spectrum of human cancer cells. In a recent study, we showed that IL-24 inhibited AKT in lung cancer cells. However, the molecular mechanism of AKT inhibition by IL-24 remains elusive.The high mobility group (HMG) A1 a member of the non-histone chromosomal proteins and commonly referred to as architectural transcription factor, regulates transcription of various genes involved in cell growth and survival. Overexpression of HMGA1 has been shown to be associated with tumor progression and metastasis in several cancers, including human lung cancer. A recent study demonstrated that HMGA1 activates AKT function by reducing the activity of the protein phosphatase, phosphatase 2A subunit B (PPP2R2A) via the oncogenic micro (mi) RNA-222. Based on this report we hypothesized that IL-24-mediated AKT inhibition involved the HMGA1/miR-222 axis.To test our hypothesis, in the present study we used a H1299 lung cancer cell line that expressed exogenous human IL-24 when induced with doxycycline (DOX). Induction of IL-24 expression in the tumor cells markedly reduced HMGA1 mRNA and protein levels. Using a mechanistic approach, we found that IL-24 reduced miR-222-3p and -5p levels, as determined by qRT-PCR. Associated with HMGA1 and miR-222 inhibition was a marked increase in PPP2R2A, with a concomitant decrease in phosphorylated AKTT308/S473 expression. SiRNA-mediated knockdown of HMGA1 in combination with IL-24 significantly reduced AKT T308/S473 protein expression and greatly reduced cell migration and invasion compared with individual treatments. Further combination of IL-24 and a miR-222-3p inhibitor significantly increased PPP2R2A expression.Our results demonstrate for the first time that IL-24 inhibits AKT via regulating the HMGA1/miR-222 signaling node in human lung cancer cells and acts as an effective tumor suppressor. Thus, a therapy combining IL-24 with HMGA1 siRNA or miR-222-3p inhibitor should present effective treatment of lung cancer.
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Proteína HMGA1a/antagonistas & inibidores , Interleucinas/fisiologia , Neoplasias Pulmonares/terapia , MicroRNAs/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular , Doxiciclina/farmacologia , Proteína HMGA1a/análise , Proteína HMGA1a/fisiologia , Humanos , MicroRNAs/fisiologia , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Interleukin (IL)-24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity.Human H1299 lung tumor cell line that was stably transfected with a doxycycline (DOX)-inducible (Tet-on) plasmid vector carrying the cDNA of IL-24-wild-type (IL-24wt) or IL-24 with all five phosphorylation sites replaced (IL-24mt) was used in the present study. Inhibition of tumor cell proliferation, cell migration and invasion, and induction of G2/M cell cycle arrest was observed in DOX-induced IL-24wt-expressing cells but not in IL-24mt-expressing cells. Secretion of IL-24mt protein was greatly reduced compared to IL-24wt protein. Further, IL-24wt and IL-24mt proteins markedly differed in their subcellular organelle localization. IL-24wt but not IL-24mt inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24wt but not IL-24mt mediated its anti-cancer activity by inhibiting the AKT signaling pathway.Our results demonstrate that IL-24 phosphorylation is required for inhibiting the AKT/mTOR signaling pathway and exerting its anti-cancer activities.
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Apoptose , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Proliferação de Células , Interleucinas/metabolismo , Neoplasias Pulmonares/patologia , Processamento de Proteína Pós-Traducional , Antibacterianos/farmacologia , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular , Doxiciclina/farmacologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Imunoprecipitação , Interleucinas/antagonistas & inibidores , Interleucinas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis and is molecular target for therapy. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated. METHODS: Human H1299, A549, H460 and HCC827 lung cancer cell lines were used in the present study. The H1299 lung cancer cell line was stably transfected with doxycycline-inducible plasmid expression vector carrying the human IL-24 cDNA and used in the present study to determine the inhibitory effects of IL-24 on SDF-1/CXCR4 axis. H1299 and A549 cell lines were used in transient transfection studies. The inhibitory effects of IL-24 on SDF1/CXCR4 and its downstream targets were analyzed by quantitative RT-PCR, western blot, luciferase reporter assay, flow cytometry and immunocytochemistry. Functional studies included cell migration and invasion assays. PRINCIPAL FINDINGS: Endogenous CXCR4 protein expression levels varied among the four human lung cancer cell lines. Doxycycline-induced IL-24 expression in the H1299-IL24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA (>40%). Functional studies showed IL-24 inhibited tumor cell migration and invasion concomitant with reduction in CXCR4 and its downstream targets (pAKTS473, pmTORS2448, pPRAS40T246 and HIF-1α). Additionally, IL-24 inhibited tumor cell migration both in the presence and absence of the CXCR4 agonist, SDF-1. Finally, IL-24 when combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration. CONCLUSIONS: IL-24 disrupts the SDF-1/CXCR4 signaling pathway and inhibits lung tumor cell migration and invasion. Additionally, IL-24, when combined with CXCR4 inhibitors exhibited enhanced anti-metastatic activity and is an attractive therapeutic strategy for lung metastasis.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Interleucinas/farmacologia , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Benzilaminas , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Ciclamos , Sinergismo Farmacológico , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Compostos Heterocíclicos/farmacologia , Humanos , Interleucinas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Cancer remains a major health issue in the world and the effectiveness of current therapies is limited resulting in disease recurrence and resistance to therapy. Therefore to overcome disease recurrence and have improved treatment efficacy there is a continued effort to develop and test new anticancer drugs that are natural or synthetic - (conventional chemotherapeutics, small molecule inhibitors) and biologic (antibody, tumor suppressor genes, oligonucleotide) product. In parallel, efforts for identifying molecular targets and signaling pathways to which cancer cells are "addicted" are underway. By inhibiting critical signaling pathways that is crucial for cancer cell survival, it is expected that the cancer cells will undergo a withdrawal symptom akin to "de-addiction" resulting in cell death. Thus, the key for having an improved and greater control on tumor growth and metastasis is to develop a therapeutic that is able to kill tumor cells efficiently by modulating critical signaling pathways on which cancer cells rely for their survival.Currently several small molecule inhibitors targeted towards unique molecular signaling pathways have been developed and tested in the clinic. Few of these inhibitors have shown efficacy while others have failed. Thus, targeting a single molecule or pathway may be insufficient to completely block cancer cell proliferation and survival. It is therefore important to identify and test an anticancer drug that can inhibit multiple signaling pathways in a cancer cell, control growth of both primary and metastatic tumors and is safe.One biologic agent that has the characteristics of serving as a potent anticancer drug is interleukin (IL)-24. IL-24 suppresses multiple signaling pathways in a broad-spectrum of human cancer cells leading to tumor cell death, inhibition of tumor angiogenesis and metastasis. Additionally, combining IL-24 with other therapies demonstrated additive to synergistic antitumor activity. Clinical testing of IL-24 as a gene-based therapeutic for the treatment of solid tumors demonstrated that IL-24 is efficacious and is safe. The unique features of IL-24 support its further development as an anticancer drug for cancer treatment.In this review we summarize the current understanding on the molecular targets and signaling pathways regulated by IL-24 in mediating its anticancer activity.