Study On The Anti-tumor Effect Of Eckol,a Brown Algae Polyphenol,and Its Mechanism

Objective:To observe the anti-tumor effect of Eckol,a brown algae polyphenol,and explore its anti-tumor mechanism,in order to provide experimental basis for the further application for Eckol in the prevention and treatment of the tumor.Methods1.Study on the anti-tumor effect of Eckol: Models of transplanted S180 ascite and sarcoma tumor-bearing mice were respectively established.Prophylactic administration of saline or Eckol(0.25 mg/kg,0.5 mg/kg,1 mg/kg)was given before modeling for 7 days.In S180 ascite tumor-bearing mice,Eckol was continuously given after modeling for 15 days.The motion status and survival days of the mice were recorded.In S180 sarcoma tumor-bearing mice,Eckol was continuously for 10 days after modeling.The body weights were recorded daily for the growth curves of mice.The mice were sacrificed at 1 hour after the last intervention,the spleen and thymus were separated for the calculation of the spleen index and thymus index;the sarcoma tissues were excised for weighing,harvested and fixed;HE staining was used to observe the morphological changes of the tumor tissues;Tumor cell apoptosis was detected by TUNEL staining;The expressions of EGFR,Bcl-2 and Caspase-3 in tumor tissue were detected by Western blot.2.Effect of Eckol on the immune function of S180 sarcoma tumor-bearing mice: Models of transplanted S180 sarcoma-bearing mice were established.Prophylactic administration was performed for 7 days before modeling and continued for 10 days after modeling.The peripheral blood serum of mice was harvested and the levels of IL-2,IL-10,TNF-?,IFN-? and TGF-? were detected by ELISA.The phagocytic function of monocyte-phagocytic cells in peripheral blood of mice was detected by carbon clearance method.Peripheral blood was collected and flow cytometry method was used to detect the ratio of T cell subsets.The spleens of mice were isolated.After smashed and filtered,the spleen cells were seeded.The transformation of spleen cells were induced by ConA.And the effect of Eckol on spleen cell transformation was measured by MTT assay.The CD11 c positive cells in tumor tissues were detected by immunohistochemistry and the infiltration of dendritic cells was observed.The mouse bone marrow-derived mononuclear cells were obtained from the bone marrow of mice and cultured by RPMI 1640 medium which contained IL-4 and GM-CSF 20 ng/ml to induce differentiation into dendritic cells.After 7 days,cells were harvested and dendritic cell phenotypes were detected by triple staining method of flow cytometry(CD11c,CD86,MHC-II).3.The effect of Eckol on Reg3A-induced over-proliferation of human SW1990 pancreatic cancer cells: SW1990 cells were cultured in vitro and treated by Eckol(0,1.25,2.5,5,10,20,40?g/ml)for 48 hours,then 50 ng/ml of exogenous Reg3 A protein was cotreated for 24 h.Cell viability was measured by MTT assay.Three concentrations of Eckol(5,10,and 20 ?g/ml)were elected and used to treat cells for 48 hours.Then,50 ng/ml of exogenous Reg3 A protein was co-treated for 24 h.Cell cycle was measured by flow cytometry;Soft agar clone formation assay was used to detect the ability of cell clone formation;Real Time PCR was used to detect the mRNA expressions of JAK2,STAT3,NF-?B and CyclinD1;Western blot was used to detect the protein expressions of JAK2/pJAK2,STAT3/p-STAT3,NF-?B,CyclinD1.Results1.Eckol had the anti-tumor effect: Eckol could extend the survival dates of S180 ascite tumor-bearing mice in vivo.In the transplanted S180 sarcoma-bearing mice model,Eckol decreased the tumor weights of sarcoma and increased the spleen indices in a dosedependent manner.Moreover,there was no significant change in the survival curves of mice.The results of HE staining showed that,compared to the model control,the microvessels in the sarcoma tissue were decreased,the levels of necrosis were increased,the nuclear pyknosis was obvious,and mononuclear cell infiltration was found around the cancer nest in the Eckol-treated groups.TUNEL staining showed that the apoptosis of tumor cells was increased in Eckol treatment groups.Western blot results showed that the expression of apoptotic protein Caspase-3 in tumor tissues was increased,and the expression of Bcl-2 and EGFR was decreased.These data suggested that Eckol has a significant antitumor effect in vivo.2.Eckol modulated the immune function of transplanted S180 sarcoma-bearing mice: ELISA results showed that Eckol could enhanced the levels of cytokines IL-2,TNF-?,IFN-? in murine serum,suggesting Eckol regulated Th1/Th2 balance,and promoted Th1-mediated cellular immunity;Eckol improved the phagocytic function of peripheral blood mononuclear phagocytic cells,promoted spleen lymphocyte transformation and regulated the proportion of peripheral blood T cell subsets in S180 sarcoma-bearing mice.Immunohistochemistry and flow cytometry showed that Eckol could promote the infiltration of dendritic cells into tumor tissues,affect the phenotype and promote the maturation of bone marrow-derived dendritic cells.These results suggested that the antitumor effect of Eckol in vivo might be related to its immunoregulatory effect on tumorbearing mice.3.Eckol inhibited the over-proliferation of SW1990 induced by Reg3 A in vitro: MTT assay showed that 5-20 ?g/ml of Eckol had no inhibitory effect on proliferation of SW1990 cells in vitro.But cell viability was decreased by the co-treatment of Eckol+Reg3A,compared to Reg3 A alone.Cell cycle assays revealed that,compared with Reg3 A alone,Eckol+Reg3A could decrease the proportion of cells in S phase and G2/M phases,and increase the proportion of cells in G0/G1 phase.Soft agar colony formation experiments showed that the number of clones in the Eckol+Reg3A group was significantly lower than that in the Reg3 A group.Real Time PCR and Western blot showed that Reg3 A up-regulated the mRNA and protein expressions of JAK2,STAT3,NF-?B and Cyclin D1,while Eckol inhibited these up-regulation effects in a concentration-dependent manner.These results suggest that Eckol could inhibit the Reg3A-induced excessive proliferation of SW1990 cells in vitro,and the mechanism might be related to JAK2/STAT3 and NF-?B/Cyclin D1 signaling pathways.Conclusion1.This study showed that Eckol had a significant anti-tumor effect in vivo,and its anti-tumor effect might be related to its immune modulatory activity in tumor-bearing mice.2.Eckol treatment in vitro inhibited the hyperproliferation of human pancreatic cancer SW1990 cells induced by Reg3 A,a pancreatic inflammation-related protein which plays an important role during pancreatic cancer progression,suggesting eckol could be investigated as a potential inhibitory agent against progression of pancreatic cancer accompanied by pancreatic inflammation.The mechanism might be related to JAK2/STAT3 and NF-?B/Cyclin D1 signaling pathways.