RESUMEN
The rBC2LCN lectin, known as a stem cell marker probe that binds to an H type 3 fucosylated trisaccharide motif, was recently revealed to also bind to pancreatic ductal adenocarcinoma (PDAC) cells. A lectin-drug conjugate was generated by fusing rBC2LCN with a cytocidal toxin, and it showed a strong anticancer effect in in vitro and in vivo PDAC models. However, it is unclear which molecules are carrier proteins of rBC2LCN on PDAC cells. In this study, we identified a rBC2LCN-positive glycoprotein expressed in PDAC. Tumor lysates of PDAC patient-derived xenografts (PDXs) were coprecipitated with rBC2LCN lectin and analyzed by liquid chromatography-mass spectrometry. A total of 343 proteins were initially identified. We used a web-based database to select five glycoproteins and independently evaluated their expression in PDAC by immunohistochemistry (IHC). Among them, we focused on carcinoembryonic antigen 5 (CEA) as the most cancer-specific carrier protein in PDAC, as it showed the most prominent difference in expression rate between PDAC cells (74%) and normal pancreatic duct cells (0%, P > .0001). rBC2LCN lectin and CEA colocalization in PDAC samples was confirmed by double-staining analysis. Furthermore, rBC2LCN-precipitated fractions were blotted with an anti-CEA polyclonal antibody (pAb), and CEA pAb-precipitated fractions were blotted with rBC2LCN lectin. The results demonstrate that CEA is in fact a ligand of rBC2LCN lectin.
Asunto(s)
Antígeno Carcinoembrionario/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proteínas Portadoras/metabolismo , Lectinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Anticuerpos/inmunología , Antígeno Carcinoembrionario/inmunología , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Femenino , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Xenoinjertos , Humanos , Inmunohistoquímica/métodos , Inmunoprecipitación/métodos , Ligandos , Ratones , Ratones Endogámicos ICR , Ratones SCID , Trasplante de Neoplasias , Neoplasias Pancreáticas/patologíaRESUMEN
Intestinal permeability is a critical factor for orally administered drugs. It can be facilitated by uptake transporters or limited by efflux transporters and metabolic enzymes in the intestine. The present study aimed to characterize the Ussing chamber system incorporating human intestinal tissue as an in vitro model for investigating the impact of intestinal uptake/efflux transporters on the intestinal absorption of substrate drugs in humans. We confirmed the functions of major intestinal uptake/efflux drug transporters in freshly isolated human jejunum sections by demonstrating a significant decrease in the mucosal uptake of cefadroxil (peptide transporter 1) and methotrexate (proton-coupled folate transporter), mucosal-to-serosal permeability of ribavirin (concentrative nucleoside transporters/equilibrative nucleoside transporters), and serosal-to-mucosal permeability of P-glycoprotein and breast cancer resistance protein substrates in the presence of their typical inhibitors. The mucosal-to-serosal apparent permeability coefficients (Papp) of 19 drugs, including substrates of drug transporters and cytochrome P450 3A, ranged from 0.60 × 10-6 to 29 × 10-6 cm/s and showed a good correlation with reported fraction of an oral dose that enters the gut wall and passes into the portal circulation with escaping intestinal metabolism (FaFg) values in humans. Furthermore, the Papp values for cefadroxil, methotrexate, and ribavirin in the presence of the corresponding transporter inhibitors underestimated the FaFg of these drugs, which clearly showed that intestinal uptake transporters facilitate their intestinal absorption in humans. In conclusion, the functions of major intestinal uptake/efflux drug transporters could be maintained in freshly isolated human jejunum sections. The Ussing chamber system incorporating human intestinal tissue would be useful for evaluating the impact of intestinal uptake/efflux transporters on the intestinal absorption of various types of drugs in humans. SIGNIFICANCE STATEMENT: Although previous studies have predicted the intestinal absorption of drugs in humans using the Ussing chamber system incorporating human intestinal tissue, there is little systematic information about drug transport mediated by multiple transporters in this system. We confirmed the functions of major intestinal uptake/efflux transporters in freshly isolated human jejunum sections and demonstrated that the mucosal-to-serosal apparent permeability coefficient of various types of drugs showed a good correlation with reported human FaFg values.
Asunto(s)
Absorción Intestinal/fisiología , Mucosa Intestinal , Yeyuno , Proteínas de Transporte de Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Administración Oral , Transporte Biológico , Humanos , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Yeyuno/patología , Circulación Hepática/fisiología , Permeabilidad , FarmacocinéticaRESUMEN
Drug resistance represents an obstacle in colorectal cancer (CRC) treatment because of its association with poor prognosis. rBC2LCN is a lectin isolated from Burkholderia that binds cell surface glycans that have fucose moieties. Because fucosylation is enhanced in many types of cancers, this lectin could be an efficient drug carrier if CRC cells specifically present such glycans. Therefore, we examined the therapeutic efficacy and toxicity of lectin drug conjugate therapy in CRC mouse xenograft models. The affinity of rBC2LCN for human CRC cell lines HT-29, LoVo, LS174T, and DLD-1 was assessed in vitro. The cytocidal efficacy of a lectin drug conjugate, rBC2LCN-38 kDa domain of pseudomonas exotoxin A (PE38) was evaluated by MTT assay. The therapeutic effects and toxicity for each CRC cell line-derived mouse xenograft model were compared between the intervention and control groups. LS174T and DLD-1 cell lines showed a strong affinity for rBC2LCN. In the xenograft model, the tumor volume in the rBC2LCN-PE38 group was significantly reduced compared with that using control treatment alone. However, the HT-29 cell line showed weak affinity and poor therapeutic efficacy. No significant toxicities or adverse responses were observed. In conclusion, we demonstrated that rBC2LCN lectin binds CRC cells and that rBC2LCN-PE38 significantly suppresses tumor growth in vivo. In addition, the efficacy of the drug conjugate correlated with its binding affinity for each CRC cell line. These results suggest that lectin drug conjugate therapy has potential as a novel targeted therapy for CRC cell surface glycans.
Asunto(s)
ADP Ribosa Transferasas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Toxinas Bacterianas/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Exotoxinas/uso terapéutico , Inmunoconjugados/uso terapéutico , Lectinas/uso terapéutico , Factores de Virulencia/uso terapéutico , ADP Ribosa Transferasas/efectos adversos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Toxinas Bacterianas/efectos adversos , Burkholderia cenocepacia/química , Línea Celular Tumoral , Supervivencia Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Portadores de Fármacos , Exotoxinas/efectos adversos , Fucosa/metabolismo , Fucosiltransferasas/metabolismo , Células HT29 , Xenoinjertos , Humanos , Inmunoconjugados/efectos adversos , Técnicas In Vitro , Lectinas/aislamiento & purificación , Lectinas/metabolismo , Ratones , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/uso terapéutico , Carga Tumoral , Factores de Virulencia/efectos adversos , Exotoxina A de Pseudomonas aeruginosaRESUMEN
INTRODUCTION: Since the outermost layer of cancer cells is covered with various glycans, targeting these groups may serve as an effective strategy in cancer therapy. We previously reported that fucosylated glycans are specifically expressed on pancreatic cancer cells, and that a protein specifically binding to these glycans, namely rBC2LCN lectin, is a potential guiding drug carrier. In the present study, a novel type of glycan-targeting nanoparticle was developed by modifying the surface of doxorubicin-containing liposomes with rBC2LCN lectin. The efficiency and specificity of this formulation, termed Lec-Doxosome, were examined in vitro and in vivo in human pancreatic cancer models. METHODS: Lec-Doxosome was prepared by a post-insertion method based on the insertion of rBC2LCN lectin into the liposomal surface via a lipid linker. The in vitro cellular binding, uptake, and cytotoxicity of Lec-Doxosome were compared with the corresponding parameters in the unmodified liposomes by applying to human pancreatic cancer cell line (Capan-1) with affinity for rBC2LCN lectin. For the in vivo assay, Lec-Doxosome was intravenously injected once per week for a total of 3 weeks into mice bearing subcutaneous tumors. RESULTS: The in vitro application of Lec-Doxosome resulted in a 1.2- to 1.6-fold higher intracellular doxorubicin accumulation and a 1.5-fold stronger cytotoxicity compared with the respective rates of accumulation and cytotoxicity in the unmodified liposomes. In vivo, Lec-Doxosome reduced the mean tumor weight (368 mg) compared with that in mice treated with unmodified liposomes (456 mg), without causing any additional adverse events. CONCLUSION: It was demonstrated from the results obtained herein that rBC2LCN lectin is a potent modifier, as a means for boosting the efficiency of nanoparticles in the targeting of cancer cell surface glycans.