Regulation of Epithelial-Mesenchymal Transition by Cyrtopodion Scabrum: An in vitro Study against Colorectal Cancer Cells.

BACKGROUND: Natural treatment of cancer has received a lot of attention recently due to its advantages including low cost, and fewer side effects. In this study, we aimed to investigate the antimetastatic properties of Cyrtopodion scabrum, a common home gecko, through Epithelial-Mesenchymal Transition (EMT) process. METHODS: Human colon cancer HCT116 cell line was selected and allocated into the following experimental groups: untreated control, vehicle control (DMSO), Retinoic acid (RA), and two treatment groups including aqueous C.scabrum Whole Extract (CWE) and C.scabrum Cell Extract (CCE) groups. The effects of the two different extracts on the viability, migration, and morphology of HCT116 cells were investigated using MTT, colony formation, and wound healing assay as well as microscopic evaluation. We also investigated the gene expression of E-cad, N-cad, and Snail genes using Real-Time PCR analysis. RESULTS: Our findings revealed that CWE and CCE were toxic to the HCT116 cell line with IC50 values of 590 and 680 µg/mL, respectively. Colony formation and migration ability of cancer cells were also inhibited by the two extracts, and the morphology of the cells were determined as epithelial phenotype. Moreover, the expression of N-cad and Snail were remarkably decreased in CWE and CCE, and RA groups, while E-cad didn't change significantly as compared to the control. CONCLUSION: The results suggest that C. scabrum extract (CsE) may induce its anti-cancer activity through the inhibition of cancer cell growth and the EMT process. CCE, as a valuable natural source, could be also suggested, to be used as an alternative/complementary medicine for the treatment of cancer, in clinical trials.

Two Simple Methods for Optimizing the Production of "Difficult-to-Express" GnRH-DFF40 Chimeric Protein.

Purpose: GnRH-DFF40 (gonadotropin releasing hormone - DNA fragmentation factor 40) is a humanized recombinant immunotoxin and serves as a prospective candidate for targeted therapy of gonadotropin releasing hormone receptor (GnRHR) overexpressing malignancies. However, its production in Escherichia coli in a soluble and functional form still remains a challenge. Here we introduce two successful and reproducible conditions for production and purification of "difficult-to-express" GnRH-DFF40 protein. Methods: A synthetic codon optimized GnRH-DFF40 fusion gene was cloned in pET28a plasmid. Two methods including high cell density IPTG induction (HCDI) and autoinduction method (AIM) with a focus on obtaining high cell density have been investigated to enhance the protein production in (E. coli). Moreover, to obtain higher protein production several factors in the AIM method including carbon sources, incubation time and temperature, plasmid stability and double colony selection, were optimized. Results: Remarkable amounts of soluble GnRH-DFF40 protein were achieved by both methods. Cell density and protein yields in AIM was about 1.5 fold higher than that what obtained using HCDI. Initial screening showed that 25ºC is better to achieve higher protein production in both methods. pH alterations in AIM were maintained in a more constant level at 25ºC and 37ºC temperatures without any detrimental effects on cell growth during protein production phase up to 21 hours after incubation. Plasmid stability during growth and expression induction phase was maintained at a high level of 98% and 96% for AIM and HCDI methods, respectively. After parameter optimization and double colony selection in AIM, a very high yield of recombinant protein was achieved (528.3 mg/L). Conclusion: With the optimization of these high cell density expression methods, reproducible manifold enhancement of soluble protein yields can be achieved for "difficult-to-express" GnRH-DFF40 compared to conventional expression methods.

The Impact of Calcium Hydroxide on the Osteoinductive Capacity of Demineralized Freeze-Dried Bone Allograft: an In-Vitro Study.

STATEMENT OF THE PROBLEM: A great challenge in periodontal therapy is the regeneration enhancement of osseous defects through applying osteoinductive materials. Demineralized freeze-dried bone allograft (DFDBA) has already been introduced as an allograft with osteoconductive and variable osteoinductive properties. Calcium hydroxide [Ca(OH)2] is an available well-known material in dentistry, which induces hard tissue formation. PURPOSE: This study evaluated the efficiency of combination of DFDBA and Ca(OH)2 in improving the quality of osteoinduction of DFDBA. MATERIALS AND METHOD: Human bone marrow-derived mesenchymal stem cells were taken from volunteers' iliac crest. Cell proliferation was determined by MTT test at 18, 24 and 48 hours post-culture in 10 groups. The employed material were 0.5, 1.0 mg/ml Ca(OH)2 in two forms of suspension and pH-adjusted solution, 10mg/ml DFDBA per se and in combination with 0.5 and 1.0 mg/ml Ca(OH)2. Mineralization was assessed by Alizarin red staining in 10 mg/ml DFDBA, DFDBA+ 0.5 and 1 mg/ml Ca(OH)2 in solution and suspension forms. The data were statistically analyzed by using one-way ANOVA and Tukey's post-hoc test (p< 0.05). RESULTS: The pH-adjusted solutions exhibited better cell proliferation compared with the suspension groups. The combination of 0.5mg/ml Ca(OH)2 solution and DFDBA increased the cell proliferation and mineralization compared with DFDBA per se (p= 0.033). CONCLUSION: The combination of Ca(OH)2 with DFDBA improved the osteoinductivity of DFDBA.