The investigation of the death-inducing potency of a recombinant Adenovector expressing Mda-7-tlyp-1 on different cancer cell lines.

Aim: The potency of Adenovector expressing Mda7-tLyp1 (Ad-Mda7-tLyp1) for death induction was evaluated on the breast (MCF7), liver (HepG2), and gastric (MKN45) cancer cell lines. Background: Mda-7 could be a possible complementary to traditional cancer therapy, and tethering to tumor-homing peptides (THPs) might improve its therapeutic efficacy. Methods: After the preparation of recombinant Ad-Mda7-tLyp1 and Ad-Mda7, the expression of recombinant proteins was analyzed by ELISA. Adenovectors were transduced (MOI=2-5) into Hep-G2, MCF7, MKN45, and normal skin fibroblast, then tumor-killing effect was measured by cytopathic effect (CPE) monitoring, MTT viability test, BAX gene expression analysis, and Caspase3/7 assay. Results: ELISA assay revealed a sustained level of recombinant protein secretion following Adenovector transduction. In CPE microscopy, all cancer cell lines showed a significant reduction (≥50%) in their normal phenotype after receiving Ad-Mda7-tLyp1 and Ad-Mda7. The viability was significantly lower compared to the control, indicating an anti-proliferating effect. In parallel, the viability test showed that Ad-Mda7 and Ad-Mda7-tLyp1 have a significant killing effect (≥50%) on MCF-7, Hep-G2, and MKN45 compared to normal fibroblast (P≤0.05). BAX gene expression analysis showed that both Ad-Mda7-tLyp1 and Ad-Mda7 vectors induced >2-fold increase of apoptosis (P<0.05), particularly in MCF7. Similarly, caspase3/7 activity showed a significant increase (P<0.05) following Ad-Mda7, and Ad-Mda7-tLyp1 transduction into cancer cell lines, but not in normal fibroblasts. Conclusion: The newly constructed Ad-Mda-tlyp1 showed a suitable tumor cell killing activity and enough specificity on studied cell lines.

The Inflammatory and Fibrotic Patterns of Hepatic Stellate Cells Following Coagulation Factors (VII or X)-Shielded Adenovirus Infection.

The role of coagulation factors on the inflammatory effect of adenovirus (Ad) is an unresolved question that was considered herein. Adenovirus-36(Ad36) and adenovector-5-GFP(Ad5-GFP) were prepared; then, they were loaded with VII or FX factors. The size/charge parameters and transduction efficiency were evaluated using fluorescent microscopy and Zetasizer, respectively. The Ad36-coagulation factor complexes were added on the stellate cells, LX-2. Thereafter, the expression levels of inflammatory and fibrotic genes including PKR, IL-1ß, TNF-α, TIMP-1, collagen, and TGF-ß were measured by qPCR and ELISA assays. The loading of FVII or FX factors not only increased the size/charge of Ad5-GFP but also enhanced the transduction rate up to 60% and 75%, respectively, compared to the controls (45%). The PKR expression analysis showed an upregulation following treatment with all Ad36 forms (P = 0.0152). The IL-1ß and TNF-α cytokines analyses demonstrated that the Ad36-FVII complex elicited the highest inflammatory response (P = 0.05). Similarly, the fibrosis-related expression analysis revealed a more inductive role of FVII when loaded on Ad36, compared to the FX factor. The findings suggested that adenovirus elicited the innate inflammatory and activation state in the hepatic stellate cell. In addition, adenovirus shielded by FVII exhibited more innate inflammation as well as activation of the stellate cells than the FX-loaded virus.

The Evaluation of tLyP-1-Bound Mda-7/IL-24 Killing Activity on a Liver Tumor Cell Line.

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.