Studies On The Effect And Mechanism Of Sulfated Polysaccharide Of Sepiella Maindroni Ink On EGFR/Akt/p38 MAPK-mediated Migration And Invasion Of Tumor Cells

ObjectivesThe morbidity and mortality of cancer patients are staying in high level,which make it urgent to discover new anticancer drug.Metastasis is the main cause of 90%of cancer associated mortality.The abnormal activation of epidermal growth factor receptor(EGFR)is related to the transformation of tumor cells from benign to malignant and the poor prognosis of cancer patients.There are many drugs targeting EGFR for cancer treatment;nevertheless,cancer cells can acquire resistance to most of these drugs.Therefore,the development of therapeutic agents blocking EGFR-mediated tumor metastasis is crucial for cancer therapy.Spiella maindroni ink polysaccharide,namely SIP,is an acidic polysaccharide isolated from the ink of cuttlefish Sepiella maindroni de Rochebruns,which has anti-mutagentic activity.After sulfation with the chlorosulfonic acid-methanamide method,the sulfated derivative of SIP,SIP-SII,was prepared.In SKOV-3 cells,SIP-SII showed significant inhibition on its migration and invasion,and induced apparent apoptosis with no cytotoxicity.Furthermore,SIP-SII could downregulate the expression of MMP-2 significantly.In vivo,SIP-SII could inhibit the growth of S180 sarcoma,the lung metastasis of B16F10 and angiogenesis.Our previous studies have demonstrated that SIP-SII possessed great potential to be anti-metastasis drug candidate.However,it is a fundermental requirement for understanding specific mechanism responsible for the anti-metastatic activity of SIP-SII before it is developed into a drug.The objectives of these studies are to dissect the mechanism of the anti-metastatic activity of SIP-SII through detecting the cellular distribution of SIP-SII and screening on the possible action target and associated signaling pathways.Methods1.The cellular distribution of SIP-SII and its effects on the expression and activation of EGFRThe SIP-SII was labeled with FITC.And then,the binding of SIP-SII to SKOV-3 cells was detected by flow cytometry.Laser scanning confocal microscope was used to detect the co-localization of SIP-SII-FITC with the specific dye for cell membrane or dye for cell nucleus.Western blot analysis was used to evaluate the effects of SIP-SII on the expression of ErbB family,including 4 different proteins,namely,EGFR,ErbB2,ErbB3 and ErbB4,as well as on the level of phosphorylated EGFR.The effect of SIP-SII on the mRNA level of EGFR was detected by RT-PCR analysis.2.The binding affinity of SIP-SII to EGFR and the effect of SIP-SII on the EGF-induced cell migration and invasionLaser scanning confocal microscope assay was used to evaluate the co-localization of SIP-SII and EGFR on cellular level,and then,SPR assay was performed to detect the binding affinity between SIP-SII and EGFR in vitro.Western blot analysis was carried out to evaluate the effects of SIP-SII on the EGF-induced EGFR auto-phosphorylation and upregulation of MMP-2 and MMP-9.The wound healing assay and matrigel invasion assay was used to detect the effect of SIP-SII on the EGF-induced tumor cell migration and invasion,repectively.3.The effects of SIP-SII on the PI3K/Akt/mTOR and MAPKs signaling pathways as well as nuclear transcription factorWestern blot assay was performed to evaluate the effect of SIP-SII on the phosphorylation level of key kinases of the PI3K/Akt/mTOR and MAPKs signaling pathways,namely,PI3K,Akt,mTOR,INK,ERK and p38 MAPK.Besides,the effects of SIP-SII on the EGF-induced phosphorylation of EGFR,Akt,JNK,ERK and p38 MAPK were detected by Western blot assay as well.Furthermore,the effects of SIP-SII on the expression of nuclear transcription factors,including NF-κB,AP-1(c-Jun and c-Fos)and AP-2 were detected using Western blot assay.The effects of SIP-SII on the EGF-induced nuclear translocation of NF-κB and AP-1 were evaluated.4.The relationship of downregulation of signaling pathways and anti-metastasis activityWound healing assay,transwell migration assay and matrigel invasion assay were used to detect whether SIP-SII inhibited the tumor cell migration and invasion via suppressing the specific signaling pathways.Western blot and RT-PCR assay were used to trace the interaction of the downregulation of MMP-2 by SIP-SII with the inhibition of specific signaling pathways.Results1.SIP-SII binds to cell membrane and inhibited the expression and phosphorylation of EGFRFITC could label the SIP-SII with low degree of substitution.SIP-SII bound to SKOV-3 cells in concentration dependent manner.Most of SIP-SII was located on the cell membrane rather than passed through it.SIP-SII only downregulated the level of EGFR protein,and had no effects on the other 3 proteins of ErbB family.Besides,SIP-SII attenuated the phosphorylation of EGFR.Furthermore,SIP-SII decreased the mRNA level of EGFR in SKOV-3 cells significantly.2.SIP-SII can bind with EGFR and attenuate the EGF-induced EGFR activation,tumor cell migration and invasion.SIP-SII was co-localizaed with EGFR in cells,and could bind with the recombinant EGFR in vitro.SIP-SII significantly inhibited the EGF-induced EGFR auto-phosphorylation in EGFR overexpressed cell lines.Moreover,SIP-SII blocked the EGF-induced upregulation of tumor cell migration,invasion and MMP-2 expression.However,SIP-SII showed no effect on the EGF-induced expression of MMP-9 in KB cells.3.SIP-SII inhibits PI3K/Akt/mTOR and p38 MAPK signaling pathways as well as nuclear transcription factor NF-κB,c-Jun,c-Fos and AP-2SIP-SII attenuated the phosphorylation level of PI3K/Akt/mTOR and p38 MAPKs signaling pathways,rather than the phosphorylation level of JNK and ERK.Furthermore,SIP-SII markedly blocked EGF-induced activation of EGFR and PI3K/Akt/mTOR and p38 MAPK.However,SIP-SII could not alter the EGF-induced upregulation of JNK and ERK.Moreover,SIP-SII decreased the expression of NF-kB,c-Jun,c-Fos and AP-2.Nevertheless,SIP-SII attenuated the EGF-induced nuclear translocation of NF-κB and c-Jun,but not c-Fos.4.SIP-SII suppresses tumor cell migration and invasion via inhibiting PI3K/Akt/mTOR and p38 MAPK signaling pathwaysSIP-SII had synergistic effect on the migration,invasion and MMP-2 expression with the specific inhibior of PI3K,mTOR and p38 MAPK.Futhermore,the specific inhibior of EGFR,P13K and p38 MAPK blocked the EGF-induced migration and invasion in KB cells,and this attenuation could be enlarged by SIP-SII.Conclusions:(1)SIP-SII binds to cell membrane and inhibits the expression and activation of EGFR,a protein expressed on cell membrane.(2)SIP-SII binds with EGFR and inhibits the EGF-induced EGFR phosphorylation,tumor cell migration and invasion as well as MMP-2 expression.(3)SIP-SII suppresses PI3K/Akt/mTOR and p38 MAPK signalng pathways,as well as the nuclear transcription factors NF-κB,AP-1 and AP-2.Moreover,SIP-SII is able to attenuate EGF-induced activation of PI3K/Akt/mTOR and p38 MAPK signalng pathways,and EGF-induced nuclear translocation of NF-κB and c-Jun.(4)SIP-SII inhibits the tumor cell migration,invasion,MMP-2 expression,EGF-induced tumor cell migration and invasion through suppressing PI3K/Akt/mTOR and p38 MAPK signalng pathways.