The Effects And Mechanisms Of Fucoidan On Invasion Of Oral Squamous Cell Carcinoma Cells And Functions Of Macrophages

BackgroundOral squamous cell carcinoma(OSCC)was one of the commonest cancers and cause of cancer-related mortality worldwide,accounting for approximately 300,400 new cases and 145,400 deaths every year.Although newly-developed therapeutic approaches showed promise,the 5-year survival rate in OSCC patients only improved 5%in the last two decades.The difficulty in treatment exists mainly due to the high tendency of local invasion and lymphatic metastasis,even in the early stage.Therefore,developing innovative therapies for reducing the invasion and metastasis potential has become a priority.Directional cell movement is largely dependent on the dynamic rearrangement of cytoskeleton,composed mainly of actin filaments,intermediate filaments and microtubules.Cytoskeletons participate in the whole process of cellular motility,provide physical force and pathways for the trafficking of motility-regulating factors.Kinesin family(KIFs)is a class of microtubule-dependent motor proteins.They play important roles in cell division and intracellular transport of vesicles by binding with microtubules.Many KIFs members adopted additional functions of regulating the dynamic assembly or disassembly of microtubules,and therefore modulate cellular motility.Our previous studies found that certain KIFs member was significantly up-regulated in primary OSCC tissue,and closely associated with lymphatic metastasis and clinical stage of patients.This result suggested that KIFs might be important regulators in the progression,invasion and metastasis of OSCC,but the specific mechanisms remain largely unknown.Tumor progression is not merely an autonomous process concerning only cancer cells,but largely modulated by the surrounding nonmalignant tissue.Tumor-infiltrated immune cells are important components of the microenvironment,and macrophages are the most abundant immunocyte population.In respond to external signals,macrophages differentiate into two commonly recognized subpopulations,anti-tumor M1 cells or tumor-promoting M2 cells.The number,phenotype and function of tumor-infiltrated macrophages are closely related with the invasive risk of OSCC.Therefore,macrophage-targeting immunotherapy might be feasible in OSCC due to its susceptibility to immunomodulation.Fucoidan is a complex of sulfated polysaccharides derived from marine brown seaweed.Fucoidan shows multiple biological activities,such as antitumor,antioxidant and inflammatory modulation,and is an excellent candidate for pharmaceuticals and nutraceuticals.Antitumor activity is a major property of fucoidan and has been well explored.Fucoidan mediated this function by not only regulating the biological behaviors of cancer cells,such as apoptosis,migration and invasion,but also modulating the activities of various types of immunocytes.However,there are no evidences about the role of fucoidan in the treatment of OSCC.In the present study,we analyzed the effects of fucoidan on invasion of tongue cancer cell line CAL27,and also its recruitment and re-education to macrophage in vitro.The specific modulatory mechanisms were also preliminary investigated.Here we found that fucoidan modified the invasion of CAL27 cells through KIF4A/MMP-2 axis,and also modified the recruitment and re-education to macrophages by regulating CCL3 production of CAL27 cells.Section I Fucoidan modulated the invasion of OSCC cells through the KIF4A/MMP-2 axisPurposeKIF members play important roles in cell movement,but their functions and modulation mechanisms in the invasion and metastasis of OSCC are not fully investigated.In this section,we explored the effects of KIF members in the invasion process of OSCC cells,and the regulation of an important natural extract,fucoidan,to KIFs expression and invasion activity of OSCC cells.Method1.Human tongue cancer cell line CAL27 was treated by different concentration of fucoidan(0,10,50,100 and 200?g/ml)for 48 h,and transwell invasion assay was performed to analyze the effect of fucoidan on invasion activity of CAL27 cells.2.CCK-8 assay was performed to analyze the effect of fucoidan(100?g/ml)on proliferation of CAL27 cells.3.qRT-PCR was performed to analyze the effect of fucoidan on matrix metalloproteinases(MMPs)expression of CAL27 cells.4.qRT-PCR was performed to analyze the effect of fucoidan on KIFs expression of CAL27 cells.5.KIF4A siRNA was transfected into CAL27 cells to knockdown its expression.Then transwell invasion assay and qRT-PCR was performed respectively to analyze the role of KIF4A in invasion and MMP-2 expression of CAL27 cells.Results1.Treatment of fucoidan(100 and 200?g/ml)for 48 h significantly reduced the invasive capacity of CAL27 cells,whereas 100?g/ml fucoidan had little effect on proliferation of CAL27 cells.2.Fucoidan treatment significantly decreased MMP-2 transcription in CAL27 cells,while the expression of MMP-9 and MMP-14 were not obviously altered.3.Fucoidan treatment significantly down-regulated the expression of KIF4A in CAL27 cells without modulating the transcriptional level of other KIF members,including KIF1B,KIF2A,KIF3A,KIF3B and KIF5B.4.KIF4A knockdown caused a decrease of MMP-2 transcription and also invasive activity of CAL27 cells.ConclusionHere we found that fucoidan decreased the invasion and MMP-2 expression in CAL27 cells,a process that dependent on KIF4A.Our findings suggested that K1F4A might become potential target for the regulation of invasive behaviors of OSCC cells.Section ? Fucoidan regulated the recruitment and re-education of macrophages by OSCC cells through CCL3PurposeMacrophages are involved in the progression of OSCC,and are potential targets for the immunotherapy of OSCC patients.Immunomodulation is an important property of fucoidan.Previous study showed that fucoidan regulated the migratory potential of macrophages.However,it is not clear the effects of fucoidan on OSCC-infiltrated macrophages.In this section,we explored the effects of fucoidan on recruitment and re-education of macrophages by OSCC cells,and preliminarily investigated the mechanisms.Methods1.THP-1-derived macrophages were differentiated in vitro.The fucoidan-treated CAL27 supernatant was collected and transwell assay was performed to testify the effects of CAL27 supernatant on recruitment of THP-1-derived macrophages.2.qRT-PCR and ELISA were performed to explore the effects of fucoidan on chemokines expression of CAL27 cells.3.CCL3 neutralizing antibody(NAb)was added into CAL27 supernatant to explore the effect of CCL3 on recruitment of THP-1-derived macrophages.4.CAL27 supernatant with/without fucoidan treatment was added during the differentiation process of THP-1-derived macrophages.qRT-PCR and ELISA were performed to testify the effects of CAL27 supernatant on cytokines expression of THP-1-derived macrophages.5.CCL3 neutralizing antibody(NAb)was added into CAL27 supernatant with/without fucoidan treatment,and then was used to stimulate THP-1-derived macrophages.qRT-PCR and ELISA were performed to testify the effects of CCL3 on cytokines expression of THP-1-derived macrophages.Results1.The CAL27 supernatant previously treated by fucoidan exhibited stronger recruiting activity to THP-1-derived macrophages.2.Fucoidan significantly increased CCL3 transcription and secretion of CAL27 cells,while showed no significant effects on production of other CCL chemokines,including CCL2,CCL4,CCL5 and CCL22.3.Blocking CCL3 in the supernatant significantly reversed the promoting effect of fucoidan on macrophages recruitment by CAL27 cells.4.Fucoidan-treated CAL27 supernatant promoted the transcription and secretion of IL-1 and TNF-a of THP-1-derived macrophages,and reduced the production of CCL22.5.CCL3 NAb significantly reversed the promoting effects of fucoidan on expression of IL-1 and TNF-? of THP-1-derived macrophages.Conclusion.We found that fucoidan promoted the recruitment of macrophages by OSCC cells,and re-educated their secretion profile toward M1-type.This function was dependent on CCL3 expression.Our findings suggested that fucoidan could modulate the local immunity status of OSCC patients,especially the number and function of tumor-infiltrated macrophages.Fucoidan might be applied as a supplementary strategy in the immunotherapy of OSCC patients.