RESUMEN
BACKGROUND: Breast cancer (BC) is one of the most common cancers in the world. Despite the many advances that have been made in treating patients, many patients are still resistant to treatment. CD44 is one of the surface glycoproteins of BC cells that plays an important role in the proliferation of these cells and inhibition of their apoptosis. Therefore, targeting it can be a treatment way for BC patients. METHODS: In this study, the effect of anti-CD44 siRNA on the proliferation, apoptosis, and migration rate of MDA-MB-231 and 4T1 cells was investigated. The techniques used in this study were MTT assay, RT-PCR, and flow cytometry. RESULTS: The apoptosis and proliferation rates in CD44 siRNA-treated cells were higher and lower, respectively, compared to untreated cells. Also, cell migration was less in treated cells compared to untreated cells. CD44 siRNA also decreased the expression of CXCR4, c-myc, Vimentin, ROCK, and MMP-9. CONCLUSION: Finally, CD44 targeting can be a good treatment option to make BC cells more sensitive to apoptosis.
Asunto(s)
Apoptosis , Neoplasias de la Mama , Receptores de Hialuranos , ARN Interferente Pequeño , Femenino , Humanos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Receptores de Hialuranos/antagonistas & inhibidores , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , ARN Interferente Pequeño/genética , Vimentina/metabolismo , Vimentina/genéticaRESUMEN
CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.
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Anticuerpos Monoclonales , Neoplasias Colorrectales , Receptores de Hialuranos , Animales , Cricetinae , Ratones , Células CHO , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/terapia , Cricetulus , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/inmunología , Isoformas de Proteínas/metabolismoRESUMEN
Background: Bone is a frequent site of metastases from breast cancer, but existing therapeutic options are not satisfactory. Although osteoblasts have active roles in cancer progression by assisting the vicious bone-destructive cycle, we employed a counterintuitive approach of activating pro-tumorigenic Wnt signaling and examined the paradoxical possibility of developing osteoblast-derived tumor-suppressive, bone-protective secretomes. Methods: Wnt signaling was activated by the overexpression of Lrp5 and ß-catenin in osteoblasts as well as a pharmacological agent (BML284), and the therapeutic effects of their conditioned medium (CM) were evaluated using in vitro cell cultures, ex vivo breast cancer tissues, and a mouse model of osteolysis. To explore the unconventional regulatory mechanism of the action of Wnt-activated osteoblasts, whole-genome proteomics analysis was conducted, followed by immunoprecipitation and gain- and loss-of-function assays. Results: While osteoblasts did not present any innate tumor-suppressing ability, we observed that the overexpression of Lrp5 and ß-catenin in Wnt signaling made their CM tumor-suppressive and bone-protective. The growth of breast cancer cells and tissues was inhibited by Lrp5-overexpressing CM (Lrp5 CM), which suppressed mammary tumors and tumor-driven bone destruction in a mouse model. Lrp5 CM also inhibited the differentiation and maturation of bone-resorbing osteoclasts by downregulating NFATc1 and cathepsin K. The overexpression of Lrp5 upregulated osteopontin that enriched Hsp90ab1 (Hsp90 beta) and moesin (MSN) in Lrp5 CM. Hsp90ab1 and MSN are atypical tumor-suppressing proteins since they are multi-tasking, moonlighting proteins that promote tumorigenesis in tumor cells. Importantly, Hsp90ab1 immuno-precipitated latent TGFß and inactivated TGFß, whereas MSN interacted with CD44, a cancer stem-cell marker, as well as fibronectin 1, an ECM protein. Furthermore, Hsp90ab1 and MSN downregulated KDM3A that demethylated histones, together with PDL1 that inhibited immune responses. Conclusion: In contrast to inducing tumor-enhancing secretomes and chemoresistance in general by inhibiting varying oncogenic pathways in chemotherapy, this study presented the unexpected outcome of generation tumor-suppressive secretomes by activating the pro-tumorigenic Wnt pathway. The results shed light on the contrasting role of oncogenic signaling in tumor cells and osteoblast-derived secretomes, suggesting a counterintuitive option for the treatment of breast cancer-associated bone metastasis.
Asunto(s)
Neoplasias de la Mama/complicaciones , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Microfilamentos/metabolismo , Osteoblastos/metabolismo , Osteólisis/prevención & control , Proteínas Supresoras de Tumor/metabolismo , Vía de Señalización Wnt , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Fibronectinas/antagonistas & inhibidores , Fibronectinas/metabolismo , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/metabolismo , Neoplasias Mamarias Experimentales/complicaciones , Neoplasias Mamarias Experimentales/terapia , Ratones , Osteoclastos/metabolismo , Osteogénesis , Osteólisis/metabolismo , Proteoma/metabolismo , Secretoma , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Radiation therapy is a current standard-of-care treatment and is used widely for GBM patients. However, radiation therapy still remains a significant barrier to getting a successful outcome due to the therapeutic resistance and tumor recurrence. Understanding the underlying mechanisms of this resistance and recurrence would provide an efficient approach for improving the therapy for GBM treatment. Here, we identified a regulatory mechanism of CD44 which induces infiltration and mesenchymal shift of GBM. Ionizing radiation (IR)-induced K-RAS/ERK signaling activation elevates CD44 expression through downregulation of miR-202 and miR-185 expression. High expression of CD44 promotes SRC activation to induce cancer stemness and EMT features of GBM cells. In this study, we demonstrate that the K-RAS/ERK/CD44 axis is a key mechanism in regulating mesenchymal shift of GBM cells after irradiation. These findings suggest that blocking the K-RAS activation or CD44 expression could provide an efficient way for GBM treatment.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Receptores de Hialuranos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Radiación Ionizante , Transducción de Señal/efectos de la radiación , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Movimiento Celular/efectos de la radiación , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/genética , Estimación de Kaplan-Meier , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Near-infrared photoimmunotherapy (NIR-PIT) is a cell selective cancer therapy that uses an antibody-photoabsorber (IRDye700DX, IR700) conjugate (APC) and NIR light. NIR-PIT targeting epidermal growth factor receptor (EGFR) in head and neck cancer (HNC) was conditionally approved in Japan in 2020. APC-bound tumors can be detected using endoscopic fluorescence imaging, whereas NIR light can be delivered using endoscopic fiber optics. The aims of this study were: (1) to assess the feasibility of endoscopic NIR-PIT in an orthotopic HNC model using a CD44-expressing MOC2-luc cell line; and (2) to evaluate quantitative fluorescence endoscopic imaging prior to and during NIR-PIT. The results were compared in 3 experimental groups: (1) untreated controls, (2) APC injection without light exposure (APC-IV), and (3) APC injection followed by NIR light exposure (NIR-PIT). APC injected groups showed significantly higher fluorescence signals for IR700 compared with the control group prior to therapeutic NIR light exposure, and the fluorescence signal significantly decreased in the NIR-PIT group after light exposure. After treatment, the NIR-PIT group showed significantly attenuated bioluminescence compared with the control and the APC-IV groups. Histology demonstrated diffuse necrotic death of the cancer cells in the NIR-PIT group alone. In conclusion, endoscopically delivered light combined with quantitative fluorescence imaging can be used to "see and treat" HNC. This method could also be applied to other types of cancer approachable with endoscopy.
Asunto(s)
Antineoplásicos Inmunológicos/administración & dosificación , Neoplasias de Cabeza y Cuello/terapia , Receptores de Hialuranos/antagonistas & inhibidores , Indoles/administración & dosificación , Compuestos de Organosilicio/administración & dosificación , Administración Intravenosa , Animales , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Endoscopía , Estudios de Factibilidad , Femenino , Neoplasias de Cabeza y Cuello/inmunología , Inmunoterapia , Indoles/química , Indoles/farmacología , Ratones , Imagen Óptica , Compuestos de Organosilicio/química , Compuestos de Organosilicio/farmacología , Fototerapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic breast cancer subtypes lacking targeted therapy. Our recent work demonstrated that circulating tumor cell (CTC) clusters and polyclonal metastasis of TNBC are driven by aggregation of CD44+ cancer stem cells (CSC) and associated with an unfavorable prognosis, such as low overall survival. However, there is no existing therapeutic that can specifically block CTC or CSC cluster formation. Methods: Using patient-derived xenograft (PDX) models, we established an ex vivo tumor cell clustering assay for a pilot screening of blockade antibodies. After identifying EGFR as a target candidate, we modulated the gene expression and inhibited its kinase activity to determine its functional importance in tumor cell clustering and therapeutic inhibition of lung metastasis. We also examined the molecular regulation network of EGFR and a potential connection to CSC marker CD44 and microRNAs, which regulate CTC clustering. Results: We report here that EGFR inhibition successfully blocks circulating CSC (cCSC) clustering and lung metastasis of TNBC. EGFR enhances CD44-mediated tumor cell aggregation and CD44 stabilizes EGFR. Importantly, blocking EGFR by a novel anti-EGFR monoclonal antibody (clone LA1) effectively blocked cell aggregation in vitro and reduced lung metastasis in vivo. Furthermore, our data demonstrated that the tumor suppressor microRNA-30c serves as another negative regulator of cCSC clustering and lung metastasis by targeting CD44 as well as its downstream effector EGFR. Conclusion: Our studies identify a novel anti-EGFR therapeutic strategy to inhibit cCSC aggregation and therefore abolish cCSC cluster-mediated metastasis of TNBC.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Agregación Celular/efectos de los fármacos , Neoplasias Pulmonares/secundario , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Antineoplásicos Inmunológicos/inmunología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Receptores ErbB/fisiología , Clorhidrato de Erlotinib/uso terapéutico , Femenino , Genes Reporteros , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/fisiología , Neoplasias Pulmonares/prevención & control , Ratones , MicroARNs/genética , Proteínas de Neoplasias/fisiología , Células Neoplásicas Circulantes/efectos de los fármacos , ARN/genética , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This work develops a new multifunctional biocompatible anticancer nanoformulation to provide targeted image-guided cancer-selective therapeutics. It consists of three active covalently bound components: (1) biocompatible nitrogen-doped graphene quantum dots (GQDs) as a multifunctional delivery and imaging platform, (2) hyaluronic acid (HA) unit targeted to the CD44 receptors on a variety of cancer cells, and (3) oxidative stress-based cancer-selective ferrocene (Fc) therapeutic. The biocompatible GQD platform synthesized from glucosamine exhibits high-yield intrinsic fluorescence. It is utilized for tracking Fc-GQD-HA formulation in vitro indicating internalization enhancement in HeLa cells targeted by the HA over non-cancer HEK-293 cells not overexpressing CD44 receptor. Fc-GQD-HA, non-toxic at 1 mg/mL to HEK-293 cells, induces cytotoxic response in HeLa enhanced over time, while therapeutic ROS generation by Fc-GQD-HA is ~3 times greater than that of Fc alone. This outlines the targeted delivery, imaging, and cancer-specific treatment capabilities of the new Fc-GQD-HA formulation enabling desired cancer-focused nanotherapeutic approach.
Asunto(s)
Sistemas de Liberación de Medicamentos , Grafito/farmacología , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Doxorrubicina/química , Doxorrubicina/farmacología , Grafito/química , Células HEK293 , Células HeLa , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/genética , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Neoplasias/genética , Neoplasias/patología , Imagen Óptica , Oxidación-Reducción/efectos de los fármacos , Puntos Cuánticos/químicaRESUMEN
CD44 is a cell surface adhesion molecule, which is overexpressed on cancer stem cells. The interaction of CD44 with hyaluronan is responsible for tumor development, metastasis, and expression of the chemoresistant phenotype. The overexpression of CD44 impedes the cytotoxic effect of chemotherapy medications in various cancers. Therefore, the high expression of CD44 is associated with a poor prognosis in affected patients. This high expression of CD44 in various cancers has provided an ample opportunity for the treatment of patients with chemoresistant malignancy. This review aims to demonstrate the various cross-talk between CD44 and intracellular and extracellular factors and highlight its role in developing chemoresistant tumors in some troublesome cancers.
Asunto(s)
Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Receptores de Hialuranos/metabolismo , Neoplasias/metabolismo , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/química , Receptores de Hialuranos/genética , Neoplasias/genética , Células Madre Neoplásicas/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. Present-day treatments have not shown real improvements in reducing the high mortality rate and the short survival of the disease. The average survival is less than 5% after 5 years. New innovative treatments are necessary to curtail the situation. The very dense pancreatic cancer stroma is a barrier that impedes the access of chemotherapeutic drugs and at the same time establishes a pro-proliferative symbiosis with the tumor, thus targeting the stroma has been suggested by many authors. No ideal drug or drug combination for this targeting has been found as yet. With this goal in mind, here we have explored a different complementary treatment based on abundant previous publications on repurposed drugs. The cell surface protein CD44 is the main receptor for hyaluronan binding. Many malignant tumors show over-expression/over-activity of both. This is particularly significant in pancreatic cancer. The independent inhibition of hyaluronan-producing cells, hyaluronan synthesis, and/or CD44 expression, has been found to decrease the tumor cell's proliferation, motility, invasion, and metastatic abilities. Targeting the hyaluronan-CD44 pathway seems to have been bypassed by conventional mainstream oncological practice. There are existing drugs that decrease the activity/expression of hyaluronan and CD44: 4-methylumbelliferone and bromelain respectively. Some drugs inhibit hyaluronan-producing cells such as pirfenidone. The association of these three drugs has never been tested either in the laboratory or in the clinical setting. We present a hypothesis, sustained by hard experimental evidence, suggesting that the simultaneous use of these nontoxic drugs can achieve synergistic or added effects in reducing invasion and metastatic potential, in PDAC. A non-toxic, low-cost scheme for inhibiting this pathway may offer an additional weapon for treating pancreatic cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Receptores de Hialuranos/genética , Hialuronano Sintasas/genética , Ácido Hialurónico/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Bromelaínas/uso terapéutico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Hialuronano Sintasas/antagonistas & inhibidores , Ácido Hialurónico/antagonistas & inhibidores , Himecromona/uso terapéutico , Terapia Molecular Dirigida , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Piridonas/farmacología , Piridonas/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
CD44 promotes metastasis, chemoresistance, and stemness in different types of cancer and is a target for the development of new anti-cancer therapies. All CD44 isoforms share a common N-terminal domain that binds to hyaluronic acid (HA). Herein, we used a computational approach to design new potential CD44 antagonists and evaluate their target-binding ability. By analyzing 30 crystal structures of the HA-binding domain (CD44HAbd), we characterized a subdomain that binds to 1,2,3,4-tetrahydroisoquinoline (THQ)-containing compounds and is adjacent to residues essential for HA interaction. By computational combinatorial chemistry (CCC), we designed 168,190 molecules and compared their conformers to a pharmacophore containing the key features of the crystallographic THQ binding mode. Approximately 0.01% of the compounds matched the pharmacophore and were analyzed by computational docking and molecular dynamics (MD). We identified two compounds, Can125 and Can159, that bound to human CD44HAbd (hCD44HAbd) in explicit-solvent MD simulations and therefore may elicit CD44 blockage. These compounds can be easily synthesized by multicomponent reactions for activity testing and their binding mode, reported here, could be helpful in the design of more potent CD44 antagonists.
Asunto(s)
Diseño de Fármacos , Descubrimiento de Drogas , Receptores de Hialuranos , Simulación de Dinámica Molecular , Tetrahidroisoquinolinas , Animales , Sitios de Unión , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/química , Ácido Hialurónico/metabolismo , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Unión Proteica , Tetrahidroisoquinolinas/químicaRESUMEN
Despite management efforts with standard surgery, radiation, and chemotherapy, glioblastoma multiform (GBM) remains resistant to treatment, which leads to tumor recurrence due to glioma stem cells (GSCs) and therapy resistance. In this study, we used random computer-based prediction and target identification to assess activities of our newly synthesized niclosamide-derived compound, NSC765689, to target GBM oncogenic signaling. Using target prediction analyses, we identified glycogen synthase kinase 3ß (GSK3ß), ß-Catenin, signal transducer and activator of transcription 3 (STAT3), and cluster of differentiation 44 (CD44) as potential druggable candidates of NSC765689. The above-mentioned signaling pathways were also predicted to be overexpressed in GBM tumor samples compared to adjacent normal samples. In addition, using bioinformatics tools, we also identified microRNA (miR)-135b as one of the most suppressed microRNAs in GBM samples, which was reported to be upregulated through inhibition of GSK3ß, and subsequently suppresses GBM tumorigenic properties and stemness. We further performed in silico molecular docking of NSC765689 with GBM oncogenes; GSK3ß, ß-Catenin, and STAT3, and the stem cell marker, CD44, to predict protein-ligand interactions. The results indicated that NSC765689 exhibited stronger binding affinities compared to its predecessor, LCC09, which was recently published by our laboratory, and was proven to inhibit GBM stemness and resistance. Moreover, we used available US National Cancer Institute (NCI) 60 human tumor cell lines to screen in vitro anticancer effects, including the anti-proliferative and cytotoxic activities of NSC765689 against GBM cells, and 50% cell growth inhibition (GI50) values ranged 0.23~5.13 µM. In summary, using computer-based predictions and target identification revealed that NSC765689 may be a potential pharmacological lead compound which can regulate GBM oncogene (GSK3ß/ß-Catenin/STAT3/CD44) signaling and upregulate the miR-135b tumor suppressor. Therefore, further in vitro and in vivo investigations will be performed to validate the efficacy of NSC765689 as a novel potential GBM therapeutic.
Asunto(s)
Biología Computacional/métodos , Glioblastoma/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Receptores de Hialuranos/antagonistas & inhibidores , Niclosamida/química , Factor de Transcripción STAT3/antagonistas & inhibidores , beta Catenina/antagonistas & inhibidores , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Diferenciación Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Simulación del Acoplamiento Molecular , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44-MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.
Asunto(s)
Hemopexina/antagonistas & inhibidores , Receptores de Hialuranos/antagonistas & inhibidores , Metaloproteinasa 9 de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Movimiento Celular , Proliferación Celular , Hemopexina/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Células MCF-7 , Metaloproteinasa 9 de la Matriz/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
CD44 is a marker of cancer stem cell (CSC) in many types of tumors. Alternative splicing of its 20 exons generates various CD44 isoforms that have different tissue specific expression and functions, including the CD44 standard isoform (CD44s) encoded by the constant exons and the CD44 variant isoforms (CD44v) with variant exon insertions. Switching between the CD44v and CD44s isoforms plays pivotal roles in tumor progression. Here we reported a novel mechanism of CD44 alternative splicing induced by TGF-ß1 and its connection to enhanced epithelial-to-mesenchymal transition (EMT) and stemness in human prostate cancer cells. TGF-ß1 treatment increased the expression of CD44s and N-cadherin while decreased the expression of CD44v and E-cadherin in DU-145 prostate cancer cells. Other EMT markers and cancer stem cell markers were also upregulated after TGF-ß1 treatment. RNAi knockdown of CD44 reversed the phenotype, which could be rescued by overexpressing CD44s but not CD44v, indicating the alternatively spliced isoform CD44s mediated the activity of TGF-ß1 treatment. Mechanistically, TGF-ß1 treatment induced the phosphorylation, poly-ubiquitination, and degradation of PCBP1, a well-characterized RNA binding protein known to regulate CD44 splicing. RNAi knockdown of PCBP1 was able to mimic TGF-ß1 treatment to increase the expression of CD44s, as well as the EMT and cancer stem cell markers. In vitro and in vivo experiments were performed to show that CD44s promoted prostate cancer cell migration, invasion, and tumor initiation. Taken together, we defined a mechanism by which TGF-ß1 induces CD44 alternative splicing and promotes prostate cancer progression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Empalme Alternativo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/genética , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Proteína smad3/antagonistas & inhibidores , Proteína smad3/genética , Proteína smad3/metabolismo , Trasplante HeterólogoRESUMEN
Anti-inflammatory drugs such as dexamethasone (DEX) are commonly administered to cancer patients along with anticancer drugs, however, the effect of DEX on human cancers is poorly understood. In this article, we have tailored self-assembled nanoparticles derived from hyaluronic acid (HA) wherein, anti-inflammatory DEX was used as a hydrophobic moiety for inducing amphiphilicity. The HA-DEX micelles were subsequently loaded with chemotherapeutic agent, doxorubicin (DOX) (HA-DEX-DOX) and was utilized to deliver drug cargo to human cancer cells expressing different levels of CD44 receptors. We found that DEX suppressed the cytotoxicity of DOX in HCT116, while it synergistically enhanced cytotoxicity in MCF-7 cells. When we tested DOX and HA-DEX-DOX in an ex-vivo human whole blood, we found activation of complement and the coagulation cascade in one group of donors. Encapsulation of DOX within the nanoparticle core eliminated such deleterious side-effects. The HA-DEX-DOX also polarized bone-marrow-derived anti-inflammatory M2 macrophages, to pro-inflammatory M1 phenotype with the upregulation of the cytokines TNF-α, iNOS and IL-1ß.
Asunto(s)
Antiinflamatorios/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Polaridad Celular/efectos de los fármacos , Dexametasona/administración & dosificación , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Ácido Hialurónico/química , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Nanopartículas/química , Animales , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Combinación de Medicamentos , Liberación de Fármacos , Células HCT116 , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Ácido Hialurónico/farmacología , Inflamación/tratamiento farmacológico , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Micelas , Fenotipo , Agregación Plaquetaria/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Systemic sclerosis a chronic, fibrotic disorder associated with high disease-specific mortality and morbidity. Cutaneous manifestations include dermal thickening and obliteration of dermal adipose tissue. Accumulation of low-molecular-weight hyaluronan, which signals through the receptor for hyaluronan-mediated motility, RHAMM, leads to progressive fibrosis and is correlated with increased severity of systemic sclerosis. The purpose of this study is to test the efficacy of two function-blocking RHAMM peptides, NPI-110 and NPI-106, in reducing skin fibrosis in a bleomycin-induced mouse model of systemic sclerosis. NPI-110 reduced visible measures of fibrosis (dermal thickness and collagen production, deposition, and organization) and profibrotic gene expression (Tgfb1, c-Myc, Col1a1, Col3a1). NPI-110 treatment also increased the expression of the antifibrotic adipokines perilipin and adiponectin. Both RHAMM peptides strongly reduced dermal RHAMM expression, predicting that dermal fibroblasts are peptide targets. Transcriptome and cell culture analyses using Rhamm-/- and Rhamm-rescued dermal fibroblasts reveal a TGFß1/RHAMM/MYC signaling axis that promotes fibrogenic gene expression and myofibroblast differentiation. RHAMM functionâblocking peptides suppress this signaling and prevent TGFß1-induced myofibroblast differentiation. These results suggest that inhibiting RHAMM signaling will offer a treatment method for cutaneous fibrosis in systemic sclerosis.
Asunto(s)
Adipoquinas/metabolismo , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Receptores de Hialuranos/antagonistas & inhibidores , Péptidos/farmacología , Esclerodermia Sistémica/tratamiento farmacológico , Piel/patología , Animales , Bleomicina/administración & dosificación , Bleomicina/toxicidad , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibrosis , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ratones , Ratones Noqueados , Péptidos/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/metabolismo , Esclerodermia Sistémica/inducido químicamente , Esclerodermia Sistémica/patología , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
CD44, as a superficial cellular glycoprotein, is an essential factor in cell-cell and cell-matrix interaction. The CD44 expression level has been substantially up-regulated in breast cancer, and this upregulation facilitates tumor proliferation and angiogenesis. This study aims to evaluate the combination therapy of Jet Pei/CD44-specific-siRNA/doxorubicin in breast cancer MDA-MB468 cell line. The MTT assay, wound healing test, colony formation assay, DAPI staining, and flow cytometry were performed to investigate the tumoral cell viability, migration, clonogenesis, and apoptosis progression. The quantitative real-time PCR (qRT-PCR) was performed to demonstrate the CD44 expression level. Finally, the effect of CD44 silencing on the expression of VEGF, CXCR4, MMP9, and MiR-142-3p was measured. The combination of CD44-specific-siRNA with doxorubicin decreased tumoral metastasis, proliferation, invasion, and migration, and increased apoptosis in MDA-MB468 cells. In conclusions, CD44 can serve as a therapeutic target in breast cancer. Moreover, the combination therapy of CD44-specific-siRNA with doxorubicin can be a promising treatment for patients with breast cancer.
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Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica , Receptores de Hialuranos/antagonistas & inhibidores , ARN Mensajero/antagonistas & inhibidores , Transfección/métodos , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Células MCF-7 , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Polietileneimina/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
For reversing the treatment failure in P-glycoprotein (P-gp)-associated MDR (multidrug resistance) of breast cancer, a high dose of Lapatinib (Lap), a substrate of breast cancer-resistant protein, was encapsulated into safe and effective acid-cleavable polysaccharide-doxorubicin (Dox) conjugates to form targeted HPP-Dox/Lap nanoparticles with an optimal drug ratio and appropriate nanosize decorated with oligomeric hyaluronic acid (HA) for specially targeting overexpressed CD44 receptors of MCF-7/ADR. The markedly increased cellular uptake and the strongest synergetic cytotoxicity revealed the enhanced reversal efficiency of HPP-Dox/Lap nanoparticles with reversal multiples at 29.83. This was also verified by the enhanced penetrating capacity in multicellular tumor spheroids. The reinforced Dox retention and substantial down-regulation of P-gp expression implied the possible mechanism of MDR reversal. Furthermore, the efficient ex vivo accumulation and distribution of nanoparticles in the tumor site and the high tumor growth inhibition (93%) even at a lower dosage (1 mg/kg) as well as lung metastasis inhibition in vivo with negligible side effects revealed the overwhelming advantages of targeted polysaccharide nanoparticles and Lap-sensitizing effect against drug-resistant tumor. The development of an efficient and nontoxic-targeted polysaccharide delivery system for reversing MDR by synergistic therapy might provide a potential clinical application value.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Lapatinib/farmacología , Nanopartículas/química , Polisacáridos/química , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Femenino , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/química , Lapatinib/química , Lapatinib/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Células MCF-7 , Ratones , Ratones Desnudos , Nanopartículas/metabolismo , Nanopartículas/toxicidad , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an antiCD44 monoclonal antibody (mAb), C44Mab5 (IgG1, kappa) was established by immunizing mice with CD44overexpressing Chinese hamster ovary (CHO)-K1 cells. C44Mab5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG1 subclass of C44Mab5 lacks antibodydependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC), the antitumor activity of C44Mab5 could not be determined. In the present study, we converted the mouse IgG1 subclass antibody C44Mab5 into an IgG2a subclass antibody, 5mG2a, and further produced a defucosylated version, 5mG2af, using FUT8deficient ExpiCHOS (BINDS09) cells. Defucosylation of 5mG2af was confirmed using fucosebinding lectins, such as AAL and PhoSL. The dissociation constants (KD) for 5mG2af against SAS and HSC2 oral cancer cells were determined through flow cytometry to be 2.8x1010 M and 2.6x109 M, respectively, indicating that 5mG2af possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5mG2af specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5mG2af showed moderate ADCC and CDC activities against SAS and HSC2 oral cancer cells. In vivo analysis revealed that 5mG2af significantly reduced tumor development in SAS and HSC2 xenografts in comparison to control mouse IgG, even after injection seven days posttumor inoculation. Collectively, these results suggest that treatment with 5mG2af may represent a useful therapy for patients with CD44expressing oral cancers.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Receptores de Hialuranos/antagonistas & inhibidores , Neoplasias de la Boca/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/aislamiento & purificación , Antineoplásicos Inmunológicos/farmacología , Células CHO , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cricetulus , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Neoplasias de la Boca/inmunología , Neoplasias de la Boca/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: Targeted multimodal approaches need to be strategically developed to control tumour growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes that arise. The tumour stage and cellular subtypes often dictate the appropriate clinical treatment regimen. Also, the development of chemoresistance is a common clinical challenge with breast cancer. Higher doses and additional drug agents can produce additional adverse effects leading to a more aggressive malignancy. Acetylsalicylic acid (ASA), metformin (Met), and oseltamivir phosphate (OP) were investigated for their efficacy to sensitize MDA-MB-231 triple-negative breast cancer and its tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR) together in combination with Tmx treatment. METHODS: Microscopic imaging, the formation of 3D multicellular tumour spheroids, immunocytochemistry, flow cytometry, Annexin V Assay, Caspase 3/7 Apoptosis Assay, tube formation assay and analysis, and WST-1 cell viability assay evaluated the formation of MCTS, morphologic changes, cell viability, apoptosis activity and the expression levels of ALDH1A1, CD44 and CD24 on the cell surface, MDA-MB231 triple-negative breast cancer, tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR). RESULTS: The results using a triple combination of ASA, Met and OP on MDA-MB-231 and MDA-MB-231-TmxR cells and their matrix-free 3D multicellular tumour spheroids (MCTS) formed by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method demonstrate a consistent and significant decrease in cell and tumour spheroid viability and volume with increased apoptotic activity, and increased sensitivity to Tmx therapy. Tmx treatment of MDA-MB-231 cells in combination with ASA, Met and OP markedly reduced the CD44/CD24 ratio by 6.5-fold compared to the untreated control group. Tmx treatment of MDA-MB-231-TmxR cells in combination with ASA, Met and OP markedly reduced the ALDH1A1 by 134-fold compared to the same treatment for the parental cell line. Also, the triple combination treatment of ASA, Met, and OP inhibited vasculogenic endothelial cell tube formation and induced endothelial cell apoptosis. CONCLUSION: For the first time, the findings demonstrate that repurposing ASA, Met, and OP provides a novel and promising targeted multimodal approach in the treatment of triple-negative breast cancer and its chemoresistant variant.
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Antineoplásicos/farmacología , Aspirina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Metformina/farmacología , Oseltamivir/farmacología , Esferoides Celulares/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Familia de Aldehído Deshidrogenasa 1/antagonistas & inhibidores , Familia de Aldehído Deshidrogenasa 1/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Antígeno CD24/antagonistas & inhibidores , Antígeno CD24/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/metabolismo , Retinal-Deshidrogenasa/antagonistas & inhibidores , Retinal-Deshidrogenasa/metabolismo , Tamoxifeno/farmacología , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/metabolismo , Células Tumorales CultivadasRESUMEN
Colorectal cancer (CRC) is one of the most common and deadly cancers in the world, mainly due to its metastatic and metabolic ability. The CD44 receptor isoform containing exon 6 (CD44v6) is a transmembrane protein that plays an important role in the establishment of tumors and metastasis, which make this molecule a potential target for therapy and diagnosis of tumors. Aiming at a targeted therapy, the anti-VEGF monoclonal antibody (mAb) bevacizumab was loaded into poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) functionalized with an antibody fragment (Fab) specific for CD44v6-expressing human cancer cells. The sizes of NPs were in the range of 150-250 nm and they had a negative charge between -5 and -10 mV, with an association efficiency (AE) of bevacizumab of 86%. v6 Fab-PLGA-PEG NPs containing bevacizumab specifically bonded to the CD44v6 cell surface receptor and exhibited higher internalization into CD44v6+ epithelial cells than bare and (-) Fab-PLGA-PEG NPs. To understand the biological effect of NP targeting, the intracellular levels of bevacizumab and VEGF were evaluated after the incubation of targeted and untargeted NPs. The intracellular levels of bevacizumab were significantly higher in cells incubated with v6 Fab-PLGA-PEG NPs and these NPs resulted in a significant decrease in the intracellular VEGF compared to untargeted NPs and free bevacizumab. PLGA-PEG NPs, surface-functionalized with a v6-specific Fab, have the potential to intracellularly deliver bevacizumab into CD44v6 expressing cancer cells.