Comparison of Inhibitory Effect of Curcumin Nanoparticles and Free Curcumin in Human Telomerase Reverse Transcriptase Gene Expression in Breast Cancer

Purpose: Telomerase is expressed in most cancers, including breast cancer. Curcumin, a polyphenolic compound that obtained from the herb of Curcuma longa, has many anticancer effects. But, its effect is low due to poor water solubility. In order to improve its solubility and drug delivery, we have utilized a β-cyclodextrin-curcumin inclusion complex. Methods: To evaluate cytotoxic effects of cyclodextrin-curcumin and free curcumin, MTT assay was done. Cells were treated with equal concentration of cyclodextrin-curcumin and free curcumin. Telomerase gene expression level in two groups was compared by Real-time PCR. Results: MTT assay demonstrated that β-cyclodextrin-curcumin enhanced curcumin delivery in T47D breast cancer cells. The level of telomerase gene expression in cells treated with cyclodextrin-curcumin was lower than that of cells treated with free curcumin (P=0.001). Conclusion: Results are suggesting that cyclodextrin-curcumin complex can be more effective than free curcumin in inhibition of telomerase expression

Inhibitory effect of gold nanoparticles conjugated with interferon gamma and methionine on breast cancer cell line

AbstractObjectiveTo develop a gold nanoparticles complex conjugated with interferon-gamma (IFN-γ) and methionine along with application of hyperthermia using near-infrared laser beams for the treatment of cancer cells.MethodsGold nanorods (10 nm) were conjugated with IFN-γ and methionine using carbodiimide family and characterized after purification by dialysis bags. Breast cancer cells were cultured and incubated with gold nanorods at different concentrations followed by irradiation with near-infrared laser beam. Samples were then evaluated for their viability in order to determine the effect of treatment and variables by MTT assy.ResultsZetasizer results confirmed the conjugation of gold nanorods with methionine and IFN-γ. The median percentage of cell viability in 0.30 μg/mL concentration of gold nanorods was 82%. The cell viability reached to 85% at the same concentration of gold nanorods, which existed in the assayed complex. The results of MTT assay showed that the 0.60 μg/mL concentration of gold nanoparticles complex was toxic on tumor cells (P < 0.05). After exposure to hyperthermia, the viability of cells at 6 min decreased to 77% in 0.30 μg/mL concentration of gold nanorods complex.ConclusionsThe size and concentration of gold nanorods was not cytotoxic. However, their presence during irradiation near-infrared laser increased the number of dead cells during the treatment of cells

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

AbstractObjectiveTo express human vascular endothelial growth factor121 (VEGF121) in insect cells.MethodsA gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF.ResultsOur results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells.ConclusionsResults from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes

Cell-specific targeting by engineered M13 bacteriophage expressing VEGFR2 nanobody

Objective(s): Filamentous bacteriophage M13 was genetically engineered to specifically target mammalian cells for gene delivery purpose. Materials and Methods: A vascular endothelial growth factor receptor 2 (VEGFR2)-specific nanobody was genetically fused to the capsid gene III of M13 bacteriophage (pHEN4/3VGR19). A mammalian expression construct containing Cop-green fluorescent protein (Cop-GFP), as a reporter gene, was amplified by PCR and then sub-cloned in the pHEN4/3VGR19 phagemid. The resulting construct was transfected into 293KDR cell. The recombinant phage was extracted and confirmed and then transduced into VEGFR2 expressing cell (293KDR). Results: Seventy-two hr after transfection, green fluorescence was detected in 30% of the cells. About 1% of the cells which transduced by recombinant phages were able to express GFP. Conclusion: It is hoped that the results from this study will help to find potential vectors to improve the efficiency of gene delivery. Taken together, we conclude that this newly-introduced vector can be used in cancer researches

Phylogenetic analysis of metalloprotease from transcriptome of venom gland of Hemiscorpius lepturus

Hemiscorpius lepturusis a dangerous scorpion and referred to health concern issue in Khuzestan, Iran. The venom of H.lepturus is cytotoxic and its effect is similar to spider Loxosceles reclusa. Metalloproteinases are the important class of enzymes in the venom that has hemorrhagic activity. The early finding suggests the existence of metalloproteases in the transcriptome of venom gland of H.lepturus. Phylogenetic analysis was accomplished to reveal the evolutionary relationship of identified metalloproteases. The phylogenetic tree was constructed by Molecular Evolutionary Genetics Analysis software and neighbor-joining method. Results showed among three sequences, two metalloproteinases named HLMP1 and HLMP3 of H.lepturus were most close to spider P. tepidariorum. The third sequence named HLMP2 was different and formed an independent clade in the phylogenetic tree. The results suggest that the sequence of metalloproteases in the venom component of H.lepturus is similar to the spider than the scorpion

Oligoclonal selection of nanobodies targeting vascular endothelial growth factor

While monoclonal antibodies are efficient therapeutics for cancer treatment, nanobodies or variable heavy domain – due to their small size, high stability, and solubility – have many advantages in comparison. Oligoclonal nanobodies are a mixture of nanobodies against different epitopes of an antigen. Specific nanobodies against vascular endothelial growth factor (VEGF, which has an important role in cancer angiogenesis) were selected from an immune camel library using biopanning. Specific binding of the nanobodies to VEGF antigen was assessed by periplasmic extract enzyme-linked immunosorbent assay (ELISA). Bioinformatics analysis and molecular docking were performed on selected nanobodies against VEGF. The in vitro inhibitory effects of each single nanobody, as well as a pool of selected nanobodies (oligoclonal nanobodies), on proliferation and tube formation by/in human umbilical vein endothelial cells (HUVEC) cells was evaluated using MTT and Tube formation assays, respectively. Four nanobodies showed the highest signal intensity in the periplasmic extract ELISA. Sequencing revealed that four unique nanobodies with different CDR3 rejoin were selected. Oligoclonal nanobodies inhibited proliferation and tube formation of the HUVEC cells more potently than did each individual nanobody. Taken together, this data from this study suggests that in vitro use of nanobodies (in an oligoclonal mode) that target distinct epitopes on VEGF could be promising as a novel therapy to treat VEGF-dependent pathologies. However, this needs to be further tested in in vivo studies

Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function

Objective(s): Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.   Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Results: Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity.  Conclusion: The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis