| Cancer is a leading cause of death worldwide, and metastasis is responsible for about90%of the deaths from cancer. Therefore, the development of antimestatic drugs is urgently needed to control human malignancies. Natural polysaccharides have become the focus of research due to the advantages such as immunoenhancing activity, antitumor activity, as well as few toxic and adverse effects. Previously, we isolated a new polysaccharide SIP from the ink of cuttlefish Sepiella maindroni. The previous studies demonstrated that sulfated SIP (SIP-S) significantly inhibited the expression as well as the activity of matrix metalloproteinase-2(MMP-2), and decreased the invasion of SKOV3cells as wll as the migration of ECV3O4cells. These results suggested that SIP-S may be a candidate compound for preventing tumor metastasis. In this study, we determined the anti-cancer and anti-metastatic activities of SIP-S in mice models, and proved the possible mechanisms of these activities.1SIP-S has anticancer and immunostimulatory activitiesThe subcutaneous transplanted model of S180sarcoma in mice was made to investigate the activities of anticancer and immunostimulatory of SIP-S.SIP-S has immunoenhancing as well as anticancer activities, and it is demonstrated that the immunoenhancing activity is one mechanism for its anticancer activity. SIP-S could significantly inhibit tumor growth in S180-bearing mice. The inhibition rates of30,20and10mg/kg·d of SIP-S were43.5%,39.7%and32.4%, respectively. There were no obvious difference between the mean tumor weight of SIP-S-treated groups and cyclophosphamide (CTX)-treated group. Besides, SIP-S could improve mice thymus and spleen index in S180-bearing mice and mice treated with CTX. The combination treatment of SIP-S with CTX showed higher anticancer potency than the monotherapy. These results showed that SIP-S has immunoenhancing and anticancer activities, and demonstrated that the immunoenhancing activity is one mechanism for SIP-S’s anticancer activity.2SIP-S could inhibit the growth and induce the apoptosis of tumor cellsSIP-S could induce apoptosis of cancer cells by regulating the expression of relevant protein, so as to inhibit the prolifieration of cancer cells.The anticancer activity of SIP-S in vitro was investigated with MTT assay and cells colony-forming experiment. The inhibition rates to SKOV3cell proliferation by incubating with500,100and20μg/mL of SIP-S for2days were41.06%,32.48%and35.10%, respectively. And, the inhibition rates to SKOV3cell colony formation with the same concenntrations of SIP-S for7days were86.96%,67.39%and50.00%, respectively. These results showed that SIP-S could inhibit tumor cells growth in vitro and the effects are associated with time and given doses.Flow cytometry combined with AnnexinV-FITC/PI staining showed that SIP-S could induce tomor cells apoptosis. The early apoptosis rates of SKOV3cells under the concentrations of500,100,20and0μg/mL for36h were8.54%,3.66%,1.79%and3.18%, respectively, and the necrosis and late apoptosis rates were17.41%,6.44%,5.76%and3.90%. Western blot analysis showed that SIP-S could suppress the expression of PARP-1and increase the expression of caspase-3,-8,-9and bax in tumor cells and the effect was associated with concentration, while it had no significant influence on the expression of bcl-2and caspase-10.3SIP-S shows anti-metastatic activity in vivo The antimetastatic activity of SIP-S in vivo was examined by using the model of artificial lung metastasis with mouse melanoma B16F10. Compared with the saline group, SIP-S (15and30mg/kg-d) administrated for12d markedly decreased B16F10pulmonary metastasis in mice models by85.94%and87.95%, respectively. Besides, SIP-S could significantly inhibit liver metastasis of B16F10and the growth of metastatic tumor. Immunohistochemistry showed that SIP-S decrease the expression levels of ICAM-1and bFGF in lung metastasis nodules.The metastatic tumor sections staining with HE showed that nuclear atypia rates in SIP-S-treated groups were decreased compared with the saline group. The expression of basic fibroblast growth factor (bFGF) and intercellular adhesion molecule-1(ICAM-1) in metastatic nodules of SIP-S-treated group was less than in that of saline group from immunohistochemical examination, which may be one of mechanisms of anti-metastatic activity of SIP-S in vivo.4SIP-S inhibits tumor cell adhesion and heparanase(HPA) activityIt is demonstrated that regulating the expression of cell adhesion molecules and inhibiting heparanase activity might be the anti-tumor metastasis mechanisms of the SIP-S.In order to explore the mechanism of anticancer activity of SIP-S, we detected the inhibitory effects of SIP-S on adhesive ability of SKOV3cells and the activity of HPA by MTT assay and enzyme-linked immunosorbent assay(ELISA assay), respectively. MTT assay showed that SIP-S could inhibit the adhesive ability of SKOV3cells to matrigel. The inhibition rate by treating with500,100and20μg/mL of SIP-S for40min were65.10%,54.40%and38.41%, while68.53%,58.27%and42.76%by treating with SIP for80min respectively. ELISA assay showed that SIP-S could significantly inhibit the degradation of HPA to its substrate heparan sulfate and the extracellular matrix. Compared with the positive control drug, the inhibition rates by treating with10and5μg/mL of SIP-S were over60%, which demonstrate SIP-S can significantly inhibit the activity of HPA. Flow cytometry assay showed that SIP-S could inhibit the P-selectin-mediated adhesion to HL-60cells. The inhibition rates with5and1mg/mL of SIP-S treatment were23.81%and20.19%, respectively. Western blot analysis showed that the expression of ICAM-1, integrin β1, TGF-β1and E-selectin in tumor cells and endothelial cells was significantly inhibited by SIP-S. But, there were no obvious change in the expression of E-cadherin and CD44V6in SKOV3cells and VCAM-1in EA.hy926cells.5SIP-S shows anti-angiogenic activityThe anti-angiogenic activity of SIP-S was detected both in vitro and in vivo. SIP-S could inhibit endothelial cell lumen formation, proven by tube formation experiments in vitro. The number of endothelial cell tube formation was significantly reduced after treatment of SIP-S500,100and20μg/mL for a few hours compared the control group. SIP-S inhibited neovascularization in chick chorioallantoic membrane (CAM) assay at the dose of2and0.4mg/mL. These results showed that SIP-S possess anti-angiogenic activity. Western blot analysis showed that SIP-S significantly decreased the expression of VEGF and bFGF in SKOV3and EA.hy926cells, which might be one of the mechanisms of anti-angiogenic effect of SIP-S.6ConclusionsIn this study, the anti-cancer and anti-metastatic effects and mechanisms of SIP-S were investigated. SIP-S has significant anti-cancer and antimetastic activities, which might be mediated by inducing tumor cell apoptosis, enhancing the immune function of tumor-bearing mice, suppressing the adhesive ability of tumor cells and inhibiting neovascularization in CAM.The success of this project will not only determine that SIP-S was a polysaccharide with good anti-cancer activity and anti-matastasis activity, but also uncover fundamental anti-metastasis mechanisms of SIP-S in vitro and in vivo.. The information we got will also improve and enhance the development of the sulfated polysaccharide based anti-cancer drug in the future. |
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