Differential Effects Of Basil Polysaccharide(BPS) And Curcumin On Migration Of SKOV3 Cells And DCs

Metastasis is the major cause of death for cancer patients. Inhibiton of invasion and migration of cancer cells may provide the effective treatment and improve the prognosis for cancer disease. Tumor invasion reflects the myriad of influences on immune competence, and several defects have been implicated. Dendritic cells (DCs) as the key antigen-presenting cells for initiating immune responses, the maturation and imgration ability of DCs are the necessary part of anti-cancer immune response. It is found that DCs are not recruited or activated in human cancers and that their function may be compromised by the tumour. Therefore, maintaince and enhensive of DCs function may provide the alternative cancer treatment.DCs function relies on migration process and they can deliver antigens in a long distance from parenchymal tissues to lymph nodes (LNs), where they can stimulate adaptive immunity. Similar with metastatic mechanism of cancer disease, MMP also play an important part in DCs migration process. Some anti-cancer drugs, such as tissue inhibitor of MMP, may also damage DCs function and impair immune response. Therefore, screening DCs stimulation adjuvants with anti-tumor effects would improve therapeutic approaches of cancer disease.Among natural biological response modifiers, polysaccharides have attracted many detailed studies about their use as immunodulators. Sweet basil (Ocimum basilicum) is commonly used in Chinese traditional medicine for detumescence, anti-inflammation and promoting circulation. Various animal models and human studies have proved that basil polysaccharide is extremely safe even at very high doses. The efficacy of basil polysaccharide in various diseases including cancer has been well established. The underlying mechanism of basil polysaccharide in anti-cancer has never been clarified. It is found that basil polysaccharide could upregulate the expression of CD80 and HLA-DR on DCs surface. Therefore, we speculate that basil polysaccharide may work as a DCs stimulation adjuvant and promote the anti-cancer effects. In this study, the potential effects and underlying mechanism of basil polysaccharide and curcumin on Human ovarian carcinoma cell lines SKOV3 and DCs were discussed, which may provide new DCs adjuvant for anti-cancer immune reponse.SubjectTo study the effects and mechanisms of basil polysaccharide(BPS) and curcumin on invasion of SKOV3 cells and the effects of both them on maturation and migration of DCs.Methods1. Generation of human monocyte-derived DCCD14+ cells from human PBMCs were enriched with a bead-labelled anti-CD14 monoclonal antibody using the magnetic antibody cell sorting (MACS) system. The purity of CD14+ monocytes was routinely over 93%. CD14+ monocytes were cultured for 5 days in complete RPMI medium containing GM-CSF (1000 U/mL), IL-4 (500 U/mL). Cells were identified as immature DCs based on the positive expression of CDla and CD209, lack of CD14 and CD83 (purity over 93%) and low expression of HLA-DR, CD80, and CD86. To induce maturation, LPS (1 μg/mL) was added on day 5, and cells were cultured for another 2 days. Cells were defined as mature DCs based on positive expression of CDla, CD209, HLA-DR, CD83 and CD86 and lack of CD 14 (purity over 93%).2. The effects of curcumin (50 μM) or BPS on DCs maturationCurcumin (50 μM), BPS (100 μg/ml) and LPS (1 μg/ml) were added imDCs and cultured for 24 hour. Expression of CD80, CD83, CD86 and HLA-DR in dendritic cells surface were analyzed by flow cytometry. Endocytic capacity of DCs were evaluated with ingesting ability of FITC-dextran. The level of IL-12 in culture supernatants from MDDCs were assayed with enzyme-linked immunosorbent assays.3. Effects and mechanisms of curcumin and basil polysaccharide (BPS) on migration of SKOV3 cells and DCsSKOV3 cells, immature or mature DCs were treated by 50 μM curcumin, or 100 μg/mL basil polysaccharide. The expression of surface receptor on SKOV-3 and DCs in the respective groups was detected using a FACSCalibur flow cytometer. Transwell assay was used to evaluate the migration ability of SKOV3 and DCs after treated with curcumin and BPS. OPN and MMP-9 in cell supernatant were measured using ELISA kits, mRNA expression of MMP-9 was analysed by quantitative reverse transcriptase-polymerase chain reaction. Western-blot method was used to evaluated the influence of curcumin and BPS on OPN expression in SKOV3 cells and DCs.Results1. Immature or mature DCs were treated with 50 uM curcumin or 100 μg/ml BPS. BPS could profoundly increased CD80, CD86, CD83 and HLA-DR expression in human monocyte-derived dendritic cells. The ability of unstimulated DC to uptake dextran was higher than that of BPS-or LPS-stimulated DC. Untreated DC secreted a low concentration of IL-12, while BPS-or LPS-stimulated DC secreted higher levels of IL-12 than untreated DC. There were no remarkable differences in the concentrations of IL-12 produced by BPS-or LPS-stimulated DC. However, curcumin effectively inhibited the surface molecules and IL-12 production induced by LPS.2. SKOV3 cells, immature or mature DCs were treated by 50 μM curcumin, or 100 μg/mL basil polysaccharide, respectively. Transwell assay demonstrated that curcumin and BPS differentially regulated the migration of SKOV3 cells and DCs: curcumin significantly decreased the migration of SKOV3 cells, immature and mature DCs, while BPS only decreased SKOV3 cells migration. Osteopontin (OPN) mRNA and protein expression were significantly reduced in curcumin and BPS-treated SKOV3 cells, and curcumin-treated DCs. Meanwhile, flow cytometry showed that curcumin significantly inhibited surface expression of CD44 in SKOV3 cells and DCs, while BPS had minimal effect on CD44 expression. The MMP-9 mRNA and protein expression were also reduced in all curcumin-treated cells and BPS-treated SKOV3 cells. The results suggested that curcumin and BPS regulated migration of SKOV3 cells and DCs by distinctly down-regulating OPN, CD44 and MMP-9 expression.Conclusion1. OPN-CD44-MMP-9 pathway may be the potential mechanism for the anti-migration ability of curcumin. BPS did not influence CD44 expression on DCs and SKOV3 cells, and other OPN receptor may get involved in the anti-migration mechanism of BPS.2. BPS induces the phenotypic maturation of PBMCs-derived DC, and curcumin partly blocked maturation in LPS-stimulated DCs.3. Curcumin impeded the migration of both SKOV3 cells and immature or mature DCs, BPS showed similar inhibitory efficiency on SKOV3 cells migration compared with curcumin, but its effect on DCs was minimal.4. Compared with curcumin, BPS seems more beneficial as a complement therapy in ovarian cancer treatment.