Synthesis and Isolation of Specific DNA Aptamer Against Ovarian CancerCell Line

  Introduction: Identification and targeting of cancer cell surface biomarkers is highly important for targeted drug delivery and reduction of chemotherapy side effect. Aptamer or chemical antibody is single-stranded DNA or RNA sequences that fold into secondary and tertiary structures making them bind to certain targets with extremely high specificity. Aptamer is a useful tool for biomarker discovery, drug targeted delivery or applied to make a biosensor. Methods and Results: In this study, the Cell-based Systematic Evolution of Liganeds by Exponential Enrichment (Cell-SELEX) was used to develop aptamer against ovarian cancer cell lines. Monitor Pool enrichment was done by flow cytometry. SSDNA of Round 12 was cloned in to pTZ57R\T vector and was sequenced. Specificity and affinity of isolated Aptamer were determined by flow cytometry... Aptamer selection was performed for 14 rounds. Round 12 selected as appropriate round for cloning. sixty aptamers were sequenced and alignment by DNAMAN software. homology of isolated aptamer was 34.1 percent. Eight aptamer were selected after phylogenic tree generated among these aptamers Mana88 sequences was specific against ovarian cancer cell line. Mana14 and Mana94 did not attached to normal cell line but they recognized other cancer cell line. Kd of isolated aptamer were 41, 250 and 2500 for Mana88, Mana14 and Mana94 respectively Conclusions: Chemotherapy is the main technique of cancer therapy; however, its side effects make it a toxic and invasive procedure. The goal of targeted chemotherapy is to overcome at least some of these nonspecific side effects. Aptamers are a class of molecule which rival antibodies in therapeutic and diagnostic applications. Mana 88 isolated in this study could use for targeted drug delivery and diagnostic ovarian cancer. Mana14 could use for targeted drug delivery ovarian and breast cancers. Isolation. Target of isolated aptamer on the cell surface will be recognized by proteomics approache

Immunological Compatibility Status of Placenta-Derived Stem Cells is Mediated by Scaffold 3D Structure

Placenta-derived amniotic epithelial cells (AECs), a great cell source for tissue engineering and stem cell therapy, are immunologically inert in their native state; however, immunological changes in these cells after culture and differentiation have challenged their applications. The aim of this study was to investigate the effect of 2D and 3D scaffolds on human lymphocyte antigens (HLA) expression by AECs. The effect of different preparation parameters including pre-freezing time and temperature was evaluated on 3D chitosan–gelatine scaffolds properties. Evaluation of MHC class I, HLA-DR and HLA-G expression in AECs after 7 d culture on 2D bed and 3D scaffold of chitosan–gelatine showed that culture of AECs on the 2D substrate up-regulated MHC class I and HLA-DR protein markers on AECs surface and down-regulated HLA-G protein. In contrast, 3D scaffold did not increase protein expression of MHC class I and HLA-DR. Moreover, HLA-G protein expression remained unchanged in 3D culture. These results confirm that 3D scaffold can remain AECs in their native immunological state and modification of physical properties of the scaffold is a key regulator of immunological markers at the gene and protein expression levels; a strategy which circumvents rejection challenge of amniotic stem cells to be translated into the clinic

Fucoxanthin Inhibits the Proliferation of ABCC2-Over Expressing Cisplatin-Resistance Ovarian Cancer Cells via Inducing Apoptosis

Background: The development of multidrug resistance (MDR) is a major barrier to achieving effective chemotherapy in cancer. Studies have shown that epithelial ovarian cancer initially responds to platinum-based therapy, however, the recurrent type is often resistant to treatment and is associated with high mortality. Fucoxanthin, a natural component found in marine algae, possesses various pharmacologic properties. This study evaluated the cytotoxicity and resistance reversal activity of fucoxanthin on multidrug resistance-associated protein 2 (MRP2)- overexpressing, cisplatin-resistant ovarian cancer cells (A2780RCIS) and their parental cells (A2780). Methods: Cell viability was evaluated in the presence of different concentrations of fucoxanthin or cisplatin or fucoxanthin/cisplatin combination using the MTT assay. Propidium iodide staining and subG1 analysis were used to evaluate fucoxanthin potential for cell cycle modification and apoptosis induction in cancer cell lines. Results: The results showed that fucoxanthin was able to cause similar toxicity in both cell lines via apoptosis induction. Co-treatment of cells with cisplatin (3.125 to 100 µM) and nontoxic concentrations of fucoxanthin (1 and 2.5 µM) did not reverse resistance to cisplatin in A2780RCIS cells. Conclusion: Although fucoxanthin was not able to modify cisplatin resistance in ovarian cancer cells, it was equally effective in inducing apoptosis and death in both A2780 and A2780RCIS cells, indicating it is not an MRP2 substrate

Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer

Objective(s): P-glycoprotein (P-gp) is an efflux protein, the overexpression of which has been associated with multidrug resistance in various cancers. Although siRNA delivery to reverse P-gp expression may be promising for sensitizing of tumor cells to cytotoxic drugs, the therapeutic use of siRNA requires effective carriers that can deliver siRNA intracellularly with minimal toxicity on target cells. We investigated a special class of PEGylated lipid-based nanoparticles (NP), named nanolipoparticles (NLPs), for siRNA- mediated P-gp downregulation. Materials and Methods: NLPs were prepared based on low detergent dialysis method. After characterization, we evaluated the effect of NLPs on siRNA delivery, and P-gp downregulation compared to oligofectamineTM (OFA) in vitro and in vivo. Results: Our results showed a significant decrease in P-gp expression and subsequent enhancement of chemosensitivity to doxorubicin in vitro. Although the effectiveness of NLPs for in vitro siRNA delivery compared to OFA was limited, the results of in vivo studies showed noticeable effectiveness of NLPs for systemic siRNA delivery. siRNA delivery using NLPs could downregulate MDR1 in tumor cells more than 80%, while OFA had a reverse effect on MDR1 expression in vivo. Conclusion: The results indicated that the prepared NLPs could be suitable siRNA delivery systems for tumor therapy

An Investigation of Innate Immune Response of Human Blood Macrophage to Sense and Antisense dsRNA

Silencing of gene expression by siRNA (small interfering RNA) is a powerful approach used to study the genetic analysis and functional roles of mammalian genes. There is at present no report about the effects of mammalian two-hybrid system plasmids delivery of sense and antisense strands. The leishmania pteridine reductase 1 (PTR1) gene was cloned as sense and antisense strands into mammalian two hybrid system plasmids. The constructs were transfected into human blood macrophages on the basis of eight experimental groups. (Antisense strand ± LPS, sense strand ± LPS, dsRNA ± LPS, negative control ± LPS). After 24 hours, cytokines production was assessed with ELISA.Transfection of sense and antisense strand RNA into monocyte-derived macrophages (MDM) was confirmed by RT-PCR. Single strands RNA expressed IL-8, IL-12, IL-1β inflammatory cytokines and dsRNA induced IL-8, IL-12 and TNF-α production in MDM. In contrast, random uptake from a mixture of two plasmids was downregulated IL-8, IL-12, IFN-γ cytokines, with a significant difference of p<0.05 in macrophage.With respect to the increased level of IL-8 in macrophage detected in single strand groups, the chemokine production—as a major feature of innate immunity—is a powerful tool for evaluation of sense/antisense in experimental and therapeutic gene vaccine delivery. siRNA–based gene therapy could have great potential in cancer treatment.Highlights siRNA (small interfering RNA) is powerful approach to study the functional roles of mammalian genes.dsRNA induced antiviral response by induction of different cytokines including TNF-α, IL-12 and IL-8.dsRNA showed promising results as a vaccine adjuvant for both antiviral and antitumor prophylaxis.The strong response of IL-8 chemokine indicated the linkage between innate immunity and adaptive immunity in progressive malignances

The Effects of Transdermal Estrogen Delivery on Bone Mineral Density in Postmenopausal Women: A Meta-analysis

Abstract Due to its minimal systematic adverse effects, transdermal estrogen is widely used for the prevention of osteoporosis in postmenopausal women. The present meta-analysis aimed to clarify the effects of transdermal estrogen on bone mineral density (BMD) of postmenopausal women. Studies were identified by searching electronic databases including Cochrane Library, MEDLINE, Embase , and CINAHL databases, and also the Sciences Citation Index. Systematic review of articles was published between January 1989 to February 2016.Reference lists of the included articles were also evaluated and consultations were made with relevant experts. While 132 studies included the desired keywords, only nine clinical trials met the inclusion criteria and were finally reviewed. The pooled percent change in BMD was statistically significant in favor of transdermal estrogen. According to resulting pooled estimate, lumbar spine BMD one and two years after transdermal estrogen therapy was respectively 3.4% (95% CI: 1.7-5.1) and 3.7% (95% CI: 1.7-5.7) higher than the baseline values. The test for heterogeneity was not statistically significant based on the I 2 heterogeneity index. One-two years of transdermal estrogen delivery can effectively increase BMD and protect the bone structure in postmenopausal women

Inhibition of Akt phosphorylation attenuates resistance to TNF-α cytotoxic effects in MCF-7 cells, but not in their doxorubicin resistant derivatives

Objective(s): Acquisition of TNF-α resistance plays role in the onset and growth of malignant tumors. Previous studies have demonstrated that MCF-7 cell line and its doxorubicin resistant variant MCF-7/Adr are resistant against the cytotoxic effects of TNF-α. In this study, we investigated the role of Akt activation in resistance of MCF-7 and MCF-7/Adr against TNF-α cytotoxicity. Materials and Methods: The role of Akt activation in TNF-α cytotoxicity was investigated by MTT cell viability assay following treatment of the cells with the chemical inhibitor of Akt activation with or without TNF-α treatment. Phosphorylation of Akt at Ser473 before and after 72 hr TNF-α treatment  was also determined by western blot. Results: TNF-α treatment led to enhancement of Akt Ser473 phosphorylation. Treatment of MCF-7 cells with TNF-α along with Akt-inhibitor agent, tricribine, attenuated Akt Ser473 phosphorylation and sensitized these cells to the cytotoxic effects of TNF-α in a dose and time dependent manner while tricribine treatment did not cause any significant cytotoxicity in MCF-7/Adr cells alone or in combination with TNF-α. Conclusion: These results demonstrate that Akt phosphorylation plays pivotal role in the resistance of MCF-7 cells against TNF-α-induced cytotoxicity while it might play no significant role in the resistance of MCF-7/Adr cells against TNF-α

Amino acid-mPEGs: Promising excipients to stabilize human growth hormone against aggregation

Objective(s): Today, the non-covalent PEGylation methods of protein pharmaceuticals attract more attention and possess several advantages over the covalent approach. In the present study, Amino Acid-mPEGs (aa-mPEGs) were synthesized, and the human Growth Hormone (hGH) stability profile was assessed in their presence and absence.Materials and Methods: aa-mPEGs were synthesized with different amino acids (Trp, Glu, Arg, Cys, and Leu) and molecular weights of polymers (2 and 5 KDa). The aa-mPEGs were analyzed with different methods. The physical and structural stabilities of hGH were analyzed by SEC and CD spectroscopy methods. Physical stability was assayed at different temperatures within certain intervals. Molecular dynamics (MD) simulation was used to realize the possible mode of interaction between protein and aa-mPEGs. The cell-based method was used to evaluate the cytotoxicity.Results: HNMR and FTIR spectroscopy indicated that aa-mPEGs were successfully synthesized. hGH as a control group is known to be stable at 4 °C; a pronounced change in monomer degradation is observed when stored at 25 °C and 37 °C. hGH:Glu-mPEG 2 kDa with a molar ratio of 1:1 to the protein solution can significantly increase the physical stability. The CD spectroscopy method showed that the secondary structure of the protein was preserved during storage. aa-mPEGs did not show any cytotoxicity activities. The results of MD simulations were in line with experimental results.Conclusion: This paper showed that aa-mPEGs are potent excipients in decreasing the aggregation of hGH. Glu-mPEG exhibited the best-stabilizing properties in a harsh environment among other aa-mPEGs

Immunogenicity and antitumor activity of the superlytic λF7 phage nanoparticles displaying a HER2/neu-derived peptide AE37 in a tumor model of BALB/c mice

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.canlet.2018.03.030 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Phage display technique has been increasingly researched for vaccine design and delivery strategies in recent years. In this study, the AE37 (Ii-Key/HER-2/neu 776–790) peptide derived from HER2 (human epidermal growth factor receptor protein) was used as a fused peptide to the lambda phage (λF7) coat protein gpD, and the phage nanoparticles were used to induce antitumor immunogenicity in a TUBO model of breast cancer in mice. Mice were immunized with the AE37 peptide displaying phage, λF7 (gpD::AE37) every 2-week intervals over 6-weeks, then the generated immune responses were evaluated. An induction of CTL immune response by the λF7 (gpD::AE37) construct compared to the control λF7 and buffer groups was observed in vitro. Moreover, in the in vivo studies, the vaccine candidate showed promising prophylactic and therapeutic effects against the HER2 overexpressing cancer in BALB/c mice.Mashhad University of Medical Sciences, Mashhad, Iran bach (MUMS GN: 922610)NSERC, Canada (NSERC GN: 214684