Self-assembled Monolayers With Different Chemical Groups As Model Substrates For Skov-3 Ovarian Cancer Cell Behaviour Studies

ObjectiveIn this study, we investigate the cell morphology, cell adhesion and progression in response to different chemical groups using the self-assembled monolayer (SAM) technique,to demonstrate the relationship between different chemical functional groups including hydroxyl (-OH), carboxyl (-COOH), animo (-NH2), mercapto (-SH) and methyl (-CH3) and ovarian cancer cells.Methods1. The SKOV-3 cells were seeded onto the SAMs surfaces that were modified with-OH,-COOH,-NH2,-CH3 or-SH chemical functional groups.2. To confirm the surface modification, the substrates with different chemical group were characterized by water contact angle measurements (Dataphysics, German).3. The morphology of the cells cultured on the modified SAMs surfaces was observed using a scanning electron microscopy (SEM, HITACHA, S-3700).4. The cells were cultured on the modified gold surfaces for 60 min,4, 6-diamidino-2-phenylindole (DAPI) staining was performed to determine the early stage adhesion of SKOV-3 cells.5. The differences of the initial cell spreading modulated by different chemical function groups were confirmed by the analysis of the focal adhesions.6. Cell proliferation was tested using the 3-diphenyltetrazolium bromide (MTT) assay at the indicated time points (5 d).7. Cell Toxicity was assessed using Live/Dead staining with Calcein-AM and PI, and lactate dehydrogenase assays.8. The apoptosis and necrosis of the SKOV-3 cells was analyzed on Guava EasyCyte 5HT flow cytometer (Millipore, USA).9. The cell cycle phase was analysed using a Guava easyCyte 5HT flow cytometer (Millipore, USA).10. Cell migration was examined by transwell migration assay.11. The expressions levels of tumor metastasis associated gene such as β-catenin and E-cadherin were analyzed by RT-PCR.Results1. The-CH3 surface was the most hydrophobic one, while-COOH and-NH2 formed moderately wettable surfaces. The-OH surface was the most hydrophilic.2. A sparse cell population and spherical cell bodies in a small area were observed with-CH3terminal group compared with the others. In contrast, cells appeared polygonal in shape and spread out to a large area on-NH2 surface, with many process contacting with each other and surfaces.3. The SKOV-3 cells cultured on-COOH and-NH2 surfaces exhibited the highest cell number during the incubation period compared with the controls (P＜0.05).4. The cell nuclei were not clearly visible, and few focal adhesions and actin stress fibres were observed on the-CH3 modified surfaces, while those on the surfaces modified with-COOH and-NH2were polygonal.5. The cells in the-CH3 culture showed the lowest proliferation (P＜0.01).6. Results of Viability/cytotoxicity staining and lactate dehydrogenase assays demonstrated that the CH3-SAMs and SH-SAMs exert cell toxicity toward Skov-3 cells.7. When cultured on the-CH3 group, approximately 12.767%of the cells were apoptotic, and approximately 12.367%were necrosis (P＜0.05).8. The percentages of the SKOV-3 cells in the mitotic phase (G2/M) when cultured on-CH3 or-OH modified surfaces were were significantly less than controls (P＜0.01).9. Cell number cultured on-CH3 surfaces was significantly lower than the control group (P＜0.01).10. The qRT-PCR results showed that the relative expression of E-cadherin and β-catenin was significantly up regulated with the-CH3 group (P＜0.01, P＜0.05), While the ratio of E-cadherin and β-catenin were both significantly down regulated in-NH2 groups (P＜0.01, P＜0.05)Conclusion1. Different chemical groups can affect Skov-3 ovarian cancer cell morphology and cell adhesion:the moderately wettable-NH2 surfaces significantly support cell adhesion; while-CH3,-SH,-OH poor cell morphology, had no obvious effect on cell adhesion.2. Different chemical groups can affect Skov-3 ovarian cancer cell proliferation, cell toxicity, cell cycle and apoptosis:CH3 inhibits the formation of actin, inhibiting cell mitosis, make ovarian cancer cell proliferation capacity decreased obviously, and has cytotoxicity, promote cell apoptosis and death;-NH2,-COOH has no cytotoxicity,-NH2 promote cell proliferation.3. Different chemical groups can affect Skov-3 ovarian cancer cell migration:-OH,-CH3 suppress oarian cancer Skov-3 cell migration ability,-NH2 promote cell migration.4. Different functional groups through the process of EMT to affect Skov-3 ovarian cancer cell biology behavior:The qRT-PCR results showed a significant down-regulation of E-cadherin and β-catenin in cells on-NH2 groups, which may caused the loss of cell properties and progression of epithelial-mesenchymal transition, promote the development of Skov-3. While The inhibitting effects of-CH3 on Skov-3 cells metastasis, were associated with the up-regulation of E-cadherin and β-catenin. CH3 may modulating the progression of Epithelial-mesenchymal transition, inhibit the invasion and metastasis of ovarian cancer cells.These results are meaningful for uncovering the interactions between the-CH3 and ovarian cancer cells, which have implications for the treatment of ovarian cancer.