RESUMEN

Although effective vaccines have been developed against SARS-CoV-2, many regions in the world still have low rates of vaccination and new variants with mutations in the viral spike protein have reduced the effectiveness of most available vaccines and treatments. There is an urgent need for a drug to cure this disease and prevent infection. The SARS-CoV-2 virus enters the host cell through protein-protein interaction between the virus's spike protein and the host's angiotensin converting enzyme (ACE2). Using protein design software and molecular dynamics simulations, we have designed a 17-residue peptide (pep39), that binds to the spike protein receptor-binding domain (RBD) and blocks interaction of spike protein with ACE2. We have confirmed the binding activity of the designed peptide for the original spike protein and the delta variant spike protein using micro-cantilever and bio-layer interferometry (BLI) based methods. We also confirmed that pep39 strongly inhibits SARS-CoV-2 virus replication in Vero E6 cells. Taken together these data suggest that a newly designed spike protein RBD blocking peptide pep39 has a potential as a SARS-CoV-2 virus inhibitor.

RESUMEN

Extracts from Euglena gracilis have been shown to prevent cancer growth in mouse models. However, the molecular mechanism of this anti-cancer activity has not been determined nor has the effect of Euglena extracts on tobacco smoke carcinogen-induced carcinogenesis. Here, we investigate the hypothesis that this anti-cancer activity is a result of changes in the intestinal microbiota induced by oral administration of the extract. We found that a Euglena gracilis water extract prevents lung tumorigenesis induced by a tobacco smoke-specific carcinogen (NNK) in mice treated either 2 weeks before or 10 weeks after NNK injection. Both of these treatment regimens are associated with significant increases in 27 microbiota metabolites found in the mouse feces, including large increases in triethanolamine, salicylate, desaminotyrosine, N-acetylserine, glycolate, and aspartate. Increases in the short-chain fatty acids (SCFAs) including acetate, propionate and butyrate are also observed. We also detected a significant attenuation of lung carcinoma cell growth through the induction of cell cycle arrest and apoptosis caused by low levels of SCFAs. This study provides strong evidence of anti-cancer activity in Euglena gracilis extracts against tobacco smoke carcinogen-induced tumorigenesis and demonstrates that this activity is linked to increased production of specific gut microbiota metabolites and the resultant induction of cell cycle arrest and apoptosis of lung carcinoma cells.

Asunto(s)

Carcinoma , Euglena gracilis , Microbioma Gastrointestinal , Neoplasias Pulmonares , Contaminación por Humo de Tabaco , Ratones , Animales , Carcinógenos/toxicidad , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/prevención & control , Contaminación por Humo de Tabaco/efectos adversos , Carcinogénesis/inducido químicamente

RESUMEN

Proteins involved in immune checkpoint pathways, such as CTLA4, PD1, and PD-L1, have become important targets for cancer immunotherapy; however, development of small molecule drugs targeting these pathways has proven difficult due to the nature of their protein-protein interfaces. Here, using a hierarchy of computational techniques, we design a cyclic peptide that binds CTLA4 and follow this with experimental verification of binding and biological activity, using bio-layer interferometry, cell culture, and a mouse tumor model. Beginning from a template excised from the X-ray structure of the CTLA4:B7-2 complex, we generate several peptide sequences using flexible docking and modeling steps. These peptides are cyclized head-to-tail to improve structural and proteolytic stability and screened using molecular dynamics simulation and MM-GBSA calculation. The standard binding free energies for shortlisted peptides are then calculated in explicit-solvent simulation using a rigorous multistep technique. The most promising peptide, cyc(EIDTVLTPTGWVAKRYS), yields the standard free energy -6.6 ± 3.5 kcal mol-1, which corresponds to a dissociation constant of ∼15 µmol L-1. The binding affinity of this peptide for CTLA4 is measured experimentally (31 ± 4 µmol L-1) using bio-layer interferometry. Treatment with this peptide inhibited tumor growth in a co-culture of Lewis lung carcinoma (LLC) cells and antigen primed T cells, as well as in mice with an orthotropic Lewis lung carcinoma allograft model.

RESUMEN

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophages), and CD4+ and CD8+ T cells, but decreased the number of LY6G+ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C.sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells.

Asunto(s)

Chlorella , Animales , Células de la Médula Ósea , Linfocitos T CD8-positivos , Pared Celular , Humanos , Lipopolisacáridos , Ratones , Bazo , Agua

RESUMEN

The antitumor effects of a partially purified water extract from Euglena gracilis (EWE) and EWE treated by boiling (bEWE) were evaluated using orthotopic lung cancer syngeneic mouse models with Lewis lung carcinoma (LLC) cells. Daily oral administration of either EWE or bEWE started three weeks prior to the inoculation of LLC cells significantly attenuated tumor growth as compared to the phosphate buffered saline (PBS) control, and the attenuation was further enhanced by bEWE. The intestinal microbiota compositions in both extract-treated groups were more diverse than that in the PBS group. Particularly, a decrease in the ratio of Firmicutes to Bacteroidetes and significant increases in Akkermansia and Muribaculum were observed in two types of EWE-treated groups. Fecal microbiota transplantation (FMT) using bEWE-treated mouse feces attenuated tumor growth to an extent equivalent to bEWE treatment, while tumor growth attenuation by bEWE was abolished by treatment with an antibiotic cocktail. These studies strongly suggest that daily oral administration of partially purified water extracts from Euglena gracilis attenuates lung carcinoma growth via the alteration of the intestinal microbiota.

Asunto(s)

Carcinoma , Euglena gracilis , Microbioma Gastrointestinal , Neoplasias Pulmonares , Administración Oral , Animales , Pulmón , Neoplasias Pulmonares/prevención & control , Ratones , Agua/farmacología

RESUMEN

A novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. In several cell culture studies, direct treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by CT26 cell antigens. In a syngeneic mouse tumor model, the growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells in immunocompetent mice. This tumor growth attenuation was further enhanced by the coadministration of the peptide form of PD-L1ip3 (10 mg/kg/day). The current study suggests that this peptide can stimulate host antitumor immunity via blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth.

RESUMEN

Beta glucans are known to have immunomodulatory effects that mediated by a variety of mechanisms. In this article, we describe experiments and simulations suggesting that beta-1,3 glucans may promote activation of T cells by a previously unknown mechanism. First, we find that treatment of a T lymphoblast cell line with beta-1,3 oligoglucan significantly increases mRNA levels of T cell activation-associated cytokines, especially in the presence of the agonistic anti-CD3 antibody. This immunostimulatory activity was observed in the absence of dectin-1, a known receptor for beta-1,3 glucans. To clarify the molecular mechanism underlying this activity, we performed a series of molecular dynamics simulations and free-energy calculations to explore the interaction of beta-1,3 oligoglucans with potential immune receptors. While the simulations reveal little association between beta-1,3 oligoglucan and the immune receptor CD3, we find that beta-1,3 oligoglucans bind to CD28 near the region identified as the binding site for its natural ligands CD80 and CD86. Using a rigorous absolute binding free-energy technique, we calculate a dissociation constant in the low millimolar range for binding of 8-mer beta-1,3 oligoglucan to this site on CD28. The simulations show this binding to be specific, as no such association is computed for alpha-1,4 oligoglucan. This study suggests that beta-1,3 glucans bind to CD28 and may stimulate T cell activation collaboratively with T cell receptor activation, thereby stimulating immune function.

Asunto(s)

Antígenos CD28/metabolismo , Activación de Linfocitos/inmunología , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , beta-Glucanos/metabolismo , Antígenos CD28/química , Citocinas/metabolismo , Humanos , Células Jurkat , Modelos Moleculares , Unión Proteica , Receptores Inmunológicos/química , Termodinámica , beta-Glucanos/química

RESUMEN

The partially purified water extract from Euglena gracilis (EWE) was evaluated for its antitumor and immunomodulatory effects in cell cultures and in a mouse orthotopic lung carcinoma allograft model. In two-dimensional cell culture, the EWE treatment inhibited cell growth of both murine Lewis lung carcinoma (LLC) and human lung carcinoma cells (A549 and H1299) in a dose- and time-dependent manner. In contrast, the growth of mouse bone marrow cells (BMCs), but not mouse splenocytes (SPLs), was stimulated by the treatment with EWE. In three-dimensional spheroid culture, spheroid growth of LLC cells was significantly attenuated by EWE treatment. In a mouse LLC orthotopic allograft model, pretreatment with EWE (150-200 mg/kg/day, via drinking water) three weeks prior to the LLC cell inoculation, but not post-treatment after LLC cell inoculation, significantly attenuated the growth of LLC tumors in immunocompetent syngeneic mouse lung. This tumor growth attenuation coincided with a significant decrease in the population of myeloid-derived cells, primarily neutrophils. Flow cytometric analysis revealed that the EWE treatment significantly attenuated growth of granulocytic myeloid-derived suppressor cells (gMDSC) in BMCs and that this decrease was due to induction of gMDSC-specific apoptosis and differentiation of monocytic MDSCs (mMDSC) to macrophages. The present study provides evidence that EWE pretreatment inhibits lung carcinoma growth mainly by stimulating host antitumor immunity through attenuation of growth of gMDSCs and decreasing the number of peripheral granulocytes. This study suggests that the partially purified extract derived from Euglena gracilis contains significant bioactive materials that prevent lung carcinoma growth.

Asunto(s)

Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Euglena gracilis/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Células A549 , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Carcinoma Pulmonar de Lewis/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/aislamiento & purificación , Factores Inmunológicos/farmacología , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Células Supresoras de Origen Mieloide/metabolismo , Factores de Tiempo , Agua/química

RESUMEN

A colon cancer growth inhibitor partially purified from the isolated cell wall membrane fraction of Chlorella sorokiniana, here referred to as Chlorella membrane factor (CMF), was evaluated for its antitumor and immunomodulatory effects in cell culture and in a colon carcinoma mouse model. The CMF treatment dose- and time-dependently inhibited colon carcinoma cell growth in 2-dimensional cultures. Treatment with CMF also significantly inhibited the growth of colon carcinoma spheroids in 3-dimensional cell culture in coculture with T lymphocytes. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of CMF (10 or 30 mg dry weight/kg body weight, every other day) dose-dependently and significantly attenuated the growth of tumor nodules via induction of tumor cell apoptosis. Evaluation of immune cell populations in ascites showed that CMF treatment tended to increase T lymphocytes but lower granulocyte populations. The present study suggests that the cell wall membrane fraction of Chlorella sorokiniana contains a bioactive material that inhibits colon carcinoma growth via direct cell growth inhibition and stimulation of host antitumor immunity. Hence, it is suggested that the Chlorella cell wall membrane extract or a bioactive substance in the extract is an attractive complementary medicine for cancer therapy.

Asunto(s)

Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Chlorella/química , Neoplasias del Colon/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Pared Celular , Colon/patología , Neoplasias del Colon/patología , Inmunidad , Inyecciones Intraperitoneales , Ratones , Extractos Vegetales/administración & dosificación

RESUMEN

Although gemcitabine is an effective chemotherapeutic for pancreatic cancer, severe side effects often accompany its use. Since we have discovered that locally administered C1B domain peptides effectively control tumor growth without any side effects, the efficacy of co-treatment with this peptide and a low dose of gemcitabine on the growth of pancreatic cancer was examined. Two- and three-dimensional cell culture studies clarified that a co-treatment with C1B5 peptide and gemcitabine significantly attenuated growth of PAN02 mouse and PANC-1 human pancreatic cancer cells in 2D and 3D cultures. Although treatment with the low dose of gemcitabine alone (76%) or the C1B5 peptide alone (39%) inhibited tumor growth moderately, a co-treatment with C1B5 peptide and a low dose of gemcitabine markedly inhibited the growth of PAN02 autografts in the mouse peritoneal cavity (94% inhibition) without any noticeable adverse effect. The number of peritoneal cavity-infiltrating neutrophils and granzyme B+ lymphocytes was significantly higher in the co-treatment group than in the control group. A significant increase of granzyme B mRNA expression was also detected in human T cells by the co-treatment. Taken together, the current study suggests that C1B5 peptide offers a remarkably effective combination treatment strategy to reduce side effects associated with gemcitabine, without losing its tumoricidal effect.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Fragmentos de Péptidos/administración & dosificación , Proteína Quinasa C/administración & dosificación , Linfocitos T/efectos de los fármacos , Animales , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Proteína Quinasa C/química , Gemcitabina

RESUMEN

The newly purified extracellular polysaccharides (exopolysaccharides) from Parachlorella kessleri (PCEPS) were evaluated on their antitumor and immunomodulatory effects in cell culture and mouse colon carcinoma peritoneal dissemination model. In two-dimensional cell culture, the PCEPS treatment inhibited cell growth of both murine and human colon carcinoma cells in a dose- and time-dependent manner. In contrast, the growth of mouse splenocytes (SPLs) and bone marrow cells (BMCs) were stimulated by the treatment with PCEPS. The treatment with PCEPS also increased specific subpopulations of the cells in BMCs: antigen presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophage) and CD8+ cytotoxic T cells. In three-dimensional spheroid culture, spheroid growth of CT26 cells co-cultured with HL-60 human neutrophilic promyeloblasts and Jurkat cells (human lymphoblasts), but not THP-1 human monocyte/macrophage was significantly attenuated by PCEPS treatment. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of PCEPS (10 mg/kg, twice per week) significantly attenuated the growth of CT26 colon carcinoma in syngeneic mice. The present study suggests that PCEPS inhibits colon carcinoma growth via direct cell growth inhibition and a stimulation of the host antitumor immune responses. Taken together, the current study suggests that exopolysaccharides derived from Parachlorella kessleri contain significant bioactive materials that inhibit colon carcinoma growth.

Asunto(s)

Antineoplásicos/uso terapéutico , Chlorella/química , Neoplasias del Colon/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Extractos Vegetales/uso terapéutico , Polisacáridos/uso terapéutico , Animales , Células de la Médula Ósea/efectos de los fármacos , Línea Celular Tumoral , Chlorophyta , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/efectos de los fármacos

RESUMEN

To evaluate the potential of cell-penetrating peptide-based delivery of apoptosis-inducer gene in cancer therapy, a modified HIV-1 TAT peptide (dimerized TAT peptide, dTAT) was studied. The dTAT and plasmid DNA (pDNA) complexes (dTAT-pDNA) were condensed using calcium chloride (dTAT-pDNA-Ca2+). This simple nonviral formulation approach showed high levels of gene expression in vitro without any cytotoxicity. In mouse studies, a single intratracheal (IT) aerosol spray or 2 intravenous (IV) injections of the dTAT, apoptosis-inducer gene, angiotensin II type 2 receptor (AT2R), and Ca2+ complexes (dTAT-pAT2R-Ca2+) significantly attenuated the acutely growing mouse Lewis lung carcinoma allografts in mouse lungs. Furthermore, single IT (p = 0.054) and the combination of IT and IV (p < 0.05) administrations of dTAT-pAT2R-Ca2+ markedly attenuated slowly growing and relatively large-sized H358 human bronchioloalveolar carcinoma xenografts in mouse lungs. These results indicate that the dTAT-pDNA-Ca2+ effectively delivered the gene to cancer cells by either IT or IV administration although the local pulmonary delivery of the dTAT-pAT2R-Ca2+ showed more effective growth inhibition of orthotopic lung cancer grafts. Thus, the present study offers preclinical proof of concept that a dTAT-based nonviral gene delivery method via IT administration may be an effective lung cancer gene therapy.

Asunto(s)

Carcinoma Pulmonar de Lewis/terapia , ADN/administración & dosificación , Técnicas de Transferencia de Gen , Neoplasias Pulmonares/terapia , Nanopartículas/química , Receptor de Angiotensina Tipo 2/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Animales , Carcinoma Pulmonar de Lewis/genética , Línea Celular Tumoral , ADN/genética , ADN/uso terapéutico , Expresión Génica , Terapia Genética , VIH-1/química , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Plásmidos/administración & dosificación , Plásmidos/genética , Plásmidos/uso terapéutico

RESUMEN

PURPOSE: To determine aerosol administration capability and therapeutic efficacy of the new formulation of hyaluronan cisplatin conjugates, HylaPlat™ (HA-Pt), for lung cancer treatment. METHODS: In vitro formulation stability test, 2D and 3D spheroid cell culture and in vivo efficacy studies using mouse orthotopic allograft models were conducted. RESULTS: The HA-Pt effectively attenuated cell growth in 2D and 3D cultures with IC50 of 2.62 and 5.36 µM, respectively, which were comparable to those with unconjugated control cisplatin-dependent growth inhibition (IC50 1.64 and 4.63 µM, respectively). A single dose of either 7.5 or 15 mg/kg HA-Pt (cisplatin equivalent) by intratracheal aerosol spray 7 days after Lewis lung carcinoma (LLC) cell inoculation markedly inhibited growth of LLC allografts in mouse lungs and resulted in a 90 or 94% reduction of tumor nodule numbers, respectively, as compared to those from the PBS control. Cancer stem cells and cisplatin resistant cells marker, CD44 expression decreased in the tumor nodules of the HA-Pt but not in those of cisplatin treated groups. CONCLUSIONS: The current study suggests that an intratracheal aerosol administration of the HA-Pt nanoparticles offers an effective strategy for lung cancer treatment and this treatment may induce only limited cisplatin resistance.

Asunto(s)

Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Ácido Hialurónico/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Nanopartículas/administración & dosificación , Tráquea/metabolismo , Células A549 , Administración por Inhalación , Animales , Antineoplásicos/metabolismo , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cisplatino/metabolismo , Vías de Administración de Medicamentos , Femenino , Humanos , Ácido Hialurónico/metabolismo , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanopartículas/metabolismo , Tráquea/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Cell-penetrating peptides (CPP) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Here, a synthetic CPP (polylysine, K9 peptide) was complexed with angiotensin II type 2 receptor (AT2R) plasmid DNA (pAT2R) and complexes were condensed using calcium chloride. The resulting complexes were small (∼150 nm) and showed high levels of gene expression in vitro and in vivo. This simple nonviral formulation approach showed negligible cytotoxicity in four different human cell lines (cervix, breast, kidney, and lung cell lines) and one mouse cell line (a lung cancer cell line). In addition, this K9-pDNA-Ca(2+) complex demonstrated cancer-targeted gene delivery when administered via intravenous injection or intratracheal spray. The transfection efficiency was evaluated in Lewis lung carcinoma (LLC) cell lines cultured in vitro and in orthotopic cancer grafts in syngeneic mice. Immunohistochemical analysis confirmed that the complex effectively delivered pAT2R to the cancer cells, where it was expressed mainly in cancer cells along with bronchial epithelial cells. A single administration of these complexes markedly attenuated lung cancer growth, offering preclinical proof-of-concept for a novel nonviral gene delivery method exhibiting effective lung tumor gene therapy via either intravenous or intratracheal administration.

Asunto(s)

Carcinoma Pulmonar de Lewis/genética , Técnicas de Transferencia de Gen , Plásmidos/genética , Polilisina , Receptor de Angiotensina Tipo 2/genética , Aloinjertos , Animales , Apoptosis/efectos de los fármacos , Calcio/química , Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Portadores de Fármacos , Expresión Génica , Terapia Genética , Humanos , Iminas/química , Inyecciones Intravenosas , Masculino , Ratones , Plásmidos/administración & dosificación , Polietilenos/química , Polilisina/química , Transfección , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analyses of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Their different tumoricidal abilities were clarified by analyzing gene expression profiles in the two types of UCMSC. Microarray analysis revealed differential gene expression between untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells. The analyses screened 17 differentially expressed genes that are commonly detected in both human and rat UCMSC. The comparison between the two sets of gene expression profiles identified two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), that were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species' breast carcinoma cells. Over-expression of FST, but not ADRP, in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. The growth of MDA-231 cells was also significantly lower when they were cultured in medium conditioned with FST, but not ADRP over-expressing human UCMSC. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-over-expressing human UCMSC significantly attenuated the tumor burden. These results suggest that FST may play an important role in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and also implies that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

Asunto(s)

Apoptosis/fisiología , Neoplasias de la Mama/fisiopatología , Proliferación Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Células Madre Mesenquimatosas/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Células Cultivadas , Análisis por Conglomerados , Femenino , Folistatina/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Perilipina-2 , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

We have recently discovered the potential involvement of angiotensin II type 2 receptor (AT2R) signaling in pancreatic cancer using AT2R deficient mice. To examine the involvement of AT2R expression in human PDAC, expressions of AT2R as well as the major angiotensin II receptor (type 1 receptor, AT1R) in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using surgically dissected human PDAC specimens. In immunohistochemical analysis, relatively strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, whereas moderate AT2R expression was detected in 78.5% of PDAC specimens and 100% of normal area of the pancreas. AT1R, but not AT2R, mRNA levels were significantly higher in the PDAC area than in the normal pancreas. AT2R mRNA levels showed a negative correlation trend with overall survival. In cell cultures, treatment with a novel AT2R agonist significantly attenuated both murine and human PDAC cell growth with negligible cytotoxicity in normal epithelial cells. In a mouse study, administrations of the AT2R agonist in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in syngeneic mice. The AT2R agonist treatment induced apoptosis primarily in tumor cells but not in stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development and pinpoint that the novel AT2R agonist could serve as an effective therapeutic for PDAC treatment.

Asunto(s)

Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Transducción de Señal , Angiotensina II/farmacología , Animales , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/agonistas , Receptor de Angiotensina Tipo 2/genética , Transducción de Señal/efectos de los fármacos , Trasplante Isogénico , Carga Tumoral/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Neoplasias Pancreáticas

RESUMEN

GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.

Asunto(s)

Angiotensina II/análogos & derivados , Antineoplásicos/química , Receptor de Angiotensina Tipo 2/agonistas , Receptor de Angiotensina Tipo 2/metabolismo , Angiotensina II/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Femenino , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/química

RESUMEN

BACKGROUND AIMS: Un-engineered human and rat umbilical cord matrix stem cells (UCMSCs) attenuate growth of several types of tumors in mice and rats. However, the mechanism by which UCMSCs attenuate tumor growth has not been studied rigorously. METHODS: The possible mechanisms of tumor growth attenuation by rat UCMSCs were studied using orthotopic Mat B III rat mammary tumor grafts in female F344 rats. Tumor-infiltrating leukocytes were identified and quantified by immunohistochemistry analysis. Potential cytokines involved in lymphocyte infiltration in the tumors were determined by microarray and Western blot analysis. The Boyden chamber migration assay was performed for the functional analysis of identified cytokines. RESULTS: Rat UCMSCs markedly attenuated tumor growth; this attenuation was accompanied by considerable lymphocyte infiltration. Immunohistochemistry analysis revealed that most infiltrating lymphocytes in the rat UCMSC-treated tumors were CD3(+) T cells. In addition, treatment with rat UCMSCs significantly increased infiltration of CD8(+) and CD4(+) T cells and natural killer (NK) cells throughout tumor tissue. CD68(+) monocytes/macrophages and Foxp3(+) regulatory T cells were scarcely observed, only in the tumors of the phosphate-buffered saline control group. Microarray analysis of rat UCMSCs demonstrated that monocyte chemotactic protein-1 is involved in rat UCMSC-induced lymphocyte infiltration in the tumor tissues. CONCLUSIONS: These results suggest that naïve rat UCMSCs attenuated mammary tumor growth at least in part by enhancing host anti-tumor immune responses. Naïve UCMSCs can be used as powerful therapeutic cells for breast cancer treatment, and monocyte chemotactic protein-1 may be a key molecule to enhance the effect of UCMSCs at the tumor site.

Asunto(s)

Tratamiento Basado en Trasplante de Células y Tejidos , Inmunidad Innata , Neoplasias Mamarias Animales/terapia , Cordón Umbilical/citología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Femenino , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/patología , Ratones , Ratas , Ratas Endogámicas F344 , Células Madre/citología , Células Madre/inmunología , Cordón Umbilical/inmunología

RESUMEN

The migratory ability of chick primordial germ cells (PGCs) transferred into quail embryos was investigated. One, ten, twenty, fifty or one hundred chick PGCs were transferred into the dorsal aorta of 2.5-day-old quail embryos. One day later, the embryos were isolated, and serial sections were prepared after embedding in paraffin. The sections were then double-stained with periodic acid-Schiff (PAS) and hematoxylin, and the numbers of PAS-positive chick PGCs in the germinal epithelium, gonadal area, head area and trunk area of the embryos were determined. Approximately 70% of the PGCs were detected in the embryos 1 day after transfer, with roughly 60% in the gonadal region and 10% in the extragonadal region. This ratio was consistent regardless of the number of PGCs transferred into the embryos. These data suggest that migration of chick PGCs into the gonadal and extragonadal regions of the quail embryo occurs probabilistically regardless of the number of chick PGCs transferred into quail embryos.

Asunto(s)

Movimiento Celular/fisiología , Coturnix/embriología , Embrión no Mamífero/fisiología , Células Germinativas/fisiología , Animales , Embrión de Pollo , Histocitoquímica/veterinaria