Coffea canephora Bean Extract Induces NIH3T3 Cell Migration

BACKGROUND: Wound healing is an essential biological process that consists of sequential steps aimed at restoring the architecture and function of damaged cells and tissues. There are empirical evidences of using pure coffee bean powder as an alternative medicine in treating various types of wounds. However, there is limited data on coffee-induced wound healing, especially migration of cells. Therefore, current study was conducted to investigate the role of coffee extract in cell migration, especially fibroblast which is important for wound healing.METHODS: Coffea canephora beans were prepared, extracted and added in the NIH3T3 cell culture in final concentration of 2.5% and 5%. Then cytotoxicity test was performed using Na,30-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) assay. Meanwhile, cell migration test was performed with scratch assay. All results were statistically analyzed.RESULTS: The 2.5% or 5% Coffea canephora beans extract (CCBE)-treated NIH3T3 cell numbers were almost similar with the numbers of NIH3T3 cells in starvation medium merely. Meanwhile, 2.5% and 5% CCBE showed significant decrease of the widths of scratched areas compared to starvation medium merely (ANOVA with LSD Post-hoc, p=0.000). After 24 h and 48 h, the average widths of 2.5% and 5% CCBE-treated scratched areas were 235.68±22.79, 50.36±5.29, 229.95±23.01, 27.68±2.83, respectively.CONCLUSION: Since both 2.5% and 5% CCBE are potential in inducing migration of fibroblast (NIH3T3 cell) and do not induce cytotoxicity, the CCBE could be potential as an agent for wound healing.KEYWORDS: coffee, Coffea canephora, NIH3T3, migration, cytotoxicity

Establishment and Application of Engineered NIH 3T3 Cell Line with Stable Human RAGE Expression

Aim NIH3T3 cell line with expression of human receptor for advanced glycation end-products (hRAGE) transduced with lentivirus vectors was used to analyze affinity, biological activity, and/or molecular mechanisms of molecules targeting the hRAGE pathway. Method The DNA fragment coding for hRAGE gene was integrated into the genome of NIH3T3 cells using lentivirus transduction. Cells expressing hRAGE were selected with puromycin, and the level of hRAGE expression was analyzed by Western blot. To establish a stable cell line, colonies of hRAGE-expressing cells were generated, and the level of RAGE expression in each engineered cell line was analyzed within 20 generations. Flow cytometry assay was used to verify affinity of anti-hRAGE antibody binding to hRAGE on the surface of engineered cells. The engineered NIH3T3 cell line was applied to assess effects of anti-hRAGE blocking antibody on amyloid β-induced cells apoptosis by CCK-8 assay. Results The engineered NIH3T3 cell line (hRAGE-NIH3T3) could stably express human RAGE. Commercial anti-RAGE polyclonal antibody could recognize and bind to human RAGE on the surface of hRAGE-NIH3T3 but not original NIH3T3 cells. In addition, hRAGE-NIH3T3 was more sensitive to RAGE pathway-dependent stimulation. Our data show that the hRAGE-NIH3T3 cell line established is an excellent tool in the study of RAGE-targeting molecules based on the cellular level, biological function, and RAGE-mediated molecular mechanisms.

CYTOTOXIC EFFECTS OF ARIPIPRAZOLE ON MKN45 AND NIH3T3 CELL LINES AND GENOTOXIC EFFECTS ON HUMAN PERIPHERAL BLOOD LYMPHOCYTES

ABSTRACT BACKGROUND: Gastric cancer is known as the fourth most common cancer. Current treatments for cancer have damaged the sensitive tissues of the healthy body, and in many cases, cancer will be recurrent. Therefore, need for treatments that are more effective is well felt. Researchers have recently shifted their attention towards antipsychotic dopamine antagonists to treat cancer. The anticancer activities of aripiprazole remain unknown. OBJECTIVE: This study aimed to evaluate the efficacy and safety of aripiprazole on gastric cancer and normal cell lines. METHODS: In this regard, the cytotoxicity and genotoxicity of aripiprazole were investigated in MKN45 and NIH3T3 cell lines by methyl tetrazolium assay and on peripheral blood lymphocytes by micronucleus assay. For this purpose, cells were cultured in 96 wells plate. Stock solutions of aripiprazole and cisplatin were prepared. After cell incubation with different concentrations of aripiprazole (1, 10, 25, 50, 100 and 200 μL), methyl tetrazolium solution was added. For micronucleus assay fresh blood was added to RPMI culture medium 1640 supplemented, and different concentrations of aripiprazole (50, 100 and 200 μL) were added. RESULTS: The finding of present study showed that the IC50 of aripiprazole in the cancer cell line (21.36 μg/mL) was lower than that in the normal cell line (54.17 μg/mL). Moreover, the micronucleus assay showed that the frequency of micronuclei of aripiprazole at concentrations below 200 μM was much less than cisplatin. CONCLUSION: Aripiprazole can be a good cytotoxic compound and good candidate for further studies of cancer therapy.

Dynamic and Depth Dependent Nanomechanical Properties of Dorsal Ruffles in Live Cells and Biopolymeric Hydrogels

The nanomechanical properties of various biological and cellular surfaces are increasingly investigated with Scanning Probe Microscopy. Surface stiffness measurements are currently being used to define metastatic properties of various cancerous cell lines and other related biological tissues. Here we present a unique methodology to understand depth dependent nanomechanical variations in stiffness in biopolymers and live cells. In this study we have used A2780 & NIH3T3 cell lines and 0.5% & 1% Agarose to investigate depth dependent stiffness and porosity on nanomechanical properties in different biological systems. This analytical methodology can circumvent the issue associated with the contribution of substrates on cell stiffness. Here we demonstrate that by calculating ‘continuous-step-wise-modulus’ on force vs. distance curves one can observe minute variation as function of depth. Due to the presence of different kinds of cytoskeletal filament, dissipation of contact force might vary from one portion of a cell to another. On NIH3T3 cell lines, stiffness profile of Circular Dorsal Ruffles could be observed in form of large parabolic feature with changes in stiffness at different depth. In biopolymers like agarose, depending upon the extent of polymerization in there can be increase or decrease in stiffness due variations in pore size and extent to which crosslinking is taking place at different depths. 0.5% agarose showed gradual decrease in stiffness whereas with 1% agarose there was slight increase in stiffness as one indents deeper into its surface.