RESUMEN
Background: The melanoma differentiation-associated gene-7 (Mda-7)/interleukin-24 (IL-24) is a tumor killing cytokine, the bystander effect of which can be enhanced through tethering to tumor homing peptides (THPs). Materials and Methods: After fusing tLyP-1, RGR, and buforin as THPs to Mda-7/IL-24, enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion potency of the recombinant proteins. The killing potency of plasmids expressing IL-24, IL-24.tLyP1, IL-24.RGR, and buf.IL-24 were assessed, using MTT, Annexin/PI staining assays, as well as measuring the expression level of GADD-153 and BCL2-associated X (BAX) on Huh-7 cells. Three-dimensional structural analysis and protein-receptor interaction were also evaluated by modeling. Results: The ELISA result showed that contrary to IL-24.RGR and buf.IL-24, IL-24.tLyP-1 retained the secretion potency, similar to the native form. The viability assessments showed that IL-24 and IL-24.tLyP-1 had the most growth suppressive effects in comparison with the control group (p < 0.0001). Furthermore, IL-24 and IL-24.tLyP-1 had the highest apoptotic activity and significant upregulatory effect on the GADD-153 and BAX genes (p < 0.0003). The modeling showed that peptide modifications left no detrimental effect on IL-24 attachment to the cognate receptor. Conclusion: IL-24 can tolerate tLyP-1 peptide modification by retaining its secretion potency. Tethering tLyP-1 to IL-24 can induce more apoptosis than its modified versions by RGR or buforin.
Asunto(s)
Apoptosis , Interleucinas/genética , Hígado/metabolismo , Péptidos Cíclicos/genética , Transfección/métodos , Línea Celular Tumoral , Supervivencia Celular , Perfilación de la Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Células Estrelladas Hepáticas/metabolismo , Humanos , Plásmidos , Proteínas Recombinantes/genética , Factor de Transcripción CHOP/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Background: Even with the fantastic successes of direct-acting antivirals (DAA) in the treatment of Hepatitis C Virus (HCV) infection, natural drug resistance remains a challenging obstacle for their impacts. The data regarding protease inhibitors (PIs) resistance in Iran population are limited. The aim of this study was to investigate the variations in NS3 protease of HCV from non-responder patients. Methods: In this cross-sectional study, 14 HCV infected patients with genotype 1(N=5) and 3(N=9) who have not responded to Interferon-related regime were enrolled from Liver Clinic, Shiraz. The NS3 protease region was amplified by Nested-PCR followed by product gel extraction. Besides, some amplified protease regions were cloned into a cloning vector to improve the sensitivity of mutation detection. Both crude and cloned sequences were then introduced into sequencing. The obtained sequences were compared with the NS3 reference sequences and analyzed by Geno2pheno available software to find possible substitutions. In the end, the phylogenetic tree was constructed. Results: Among variations responsible for PIs resistance, only one out of 14 (7%) sample who was infected with genotype 1a, harbored R117C+N174S double mutation, which causes reduced susceptibility to Telaprevir. Any another resistance mutation was not found among the studied population. The most frequent substitutions were determined as I52M(N=9), S102A(N=9), S166A(8) and V170I(8) for genotype 3a, and F147S/A(4) for genotype 1. However, some uncharacterized substitutions on scored position, including I132L(N=1), I170V(N=3) and N174S(N=2) were also determined among sequences. Phylogenetic analysis demonstrated that the protease region has enough power to correctly classify enrolled samples into relevant clusters on the tree. There were 2, 3 and 9 cases of sub-genotypes 1a, 1b, and 3a, respectively. Conclusion: A low frequency of PIs resistance mutations in our HCV infected population is a hopeful point of starting these drugs in HCV infected patients.
Asunto(s)
Farmacorresistencia Viral/genética , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Mutación , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/genética , Adulto , Anciano , Sustitución de Aminoácidos , Antivirales/farmacología , Estudios Transversales , Femenino , Estudios de Seguimiento , Hepacivirus/enzimología , Hepacivirus/genética , Hepatitis C/virología , Humanos , Masculino , Persona de Mediana Edad , FilogeniaRESUMEN
INTRODUCTION: MDA-7/Interleukin-24 (IL-24), as a pleiotropic cytokine, exhibits a specific tumor suppression property that has attracted a great deal of attention. While its anti-tumor induction is mostly attributed to endogenous gene expression, attachment of secreted MDA-7/IL-24 to cognate receptors also triggers the death of cancerous cell via different pathways. Therefore, precise targeting of secreted MDA-7/IL-24 to tumor cells would render it more efficacy and specificity. AREAS COVERED: In order to target soluble cytokines, particularly MDA-7/IL-24 to the neighbor tumor sites and enhance their therapeutic efficiency, fusing with cell penetrating peptides (CPPs) or Tumor homing peptides (THPs) seems logical due to the improvement of their bystander effects. Although the detailed anti-tumor mechanisms of endogenous mda-7/IL-24 have been largely investigated, the significance of the secreted form in these activities and methods of its improving by CPPs or THPs need more discussion. EXPERT OPINION: While the employment of CPPs/THPs for the improvement of cytokine gene therapy is desirable, to create fusions of CPPs/THPs with MDA-7/IL-24, some hurdles are not avoidable. Regarding our expertise, herein, the importance of CPPs/THPs, needs for their elegant designing in a fusion structure, and their applications in cytokine gene therapy are discussed with a special focus on mda-7/IL-24.