Antitumor Effects And Mechanisms Of Laryngeal Squamous Cell Carcinoma By Combination Of Oridonin And Cetuximab

Laryngeal cancer is a common type of head and neck malignancy worldwide.Laryngeal cancer accounts for about 1%-5%of all malignant tumors.Most laryngeal cancers have been pathologically identified as laryngeal squamous cell carcinoma(LSCC).Epidermal growth factor receptor(EGFR).a transmembrane glycoprotein,is expressed at high levels in 90%cases of patients with LSCC.Cetuximab(Cet),an anti-EGFR monoclonal antibody,was approved for the treatment of head and neck cancers by the United States Food and Drug Administration(USFDA).However,the antitumor efficacy of Cet was less than 15%in head and neck squamous cell carcinoma(HNSCC)patients.Therefore,the combination with Cet and anti-cancer agents might be an effective management for LSCC.Oridonin(ORI)is a natural and safe kaurene diterpenoid isolated from traditional Chinese medicine Rabdosia ruhescens.Several reports have proved that ORI inhibits the growth of tumor cell lines in vitro and transplanted animal tumors.which are associated with the inhibition of cell proliferation.the induction of apoptosis and autophagy,and so on.Our previous studies showed that ORI inhibited cell growth in HEp-2 cells through inhibition of EGFR.In this study.ORI significantly inhibited overexpression EGFR LSCC cell growth,while ORI showed less effective in the growth inhibition of human breast cancer MCF-7 cells,human cervical carcinoma Hela cells and non-small cell lung cancer A549 cells.In addition,the AutoDock molecular docking software was used to evaluate the binding of ORI and EGFR.The result showed that the binding affinity is-8.34 kcal/mol and ORI could form several hydrogen bonds and hydrophobic interactions with the amino acid residues of EGFR.The results of Autodock and experiments proved that ORI could inhibit the proliferation of LSCC cells through strongly target to EGFR.Therefore,the in vitro and in vivo experiments were used to investigate the antitumor effect and related mechanism of combination of Cet and ORI.In this study,to investigate whether ORI could improve the anticancer efficacy of Cet,two LSCC cell lines HEp-2 and Tu212 cells were used to evaluate the effect of ORI,Cet alone and the combination on the cell proliferation and EGFR signaling pathway.The in vitro results showed that the combination treatment with Cet and ORI synergistically inhibited cell growth in LSCC cells through dual inhibition of EGFR signaling pathway.1h pretreatment ORI with Cet showed stronger effect in killing LSCC cells.Moreover,in nude mice bearing HEp-2 xenografts,ORI plus Cet caused a more significant tumor regression compared with single agents.Western blotting and immunohistochemistry showed that ORI plus Cet significantly downregulated the levels of proliferating cell nuclear antigen(PCNA)and p-EGFR in tumor tissues,indicating that combination of ORI with Cet inhibited LSCC cells proliferation through inhibiting EGFR pathway.We further investigated the mechanisms of cell death induced by combination of ORI with Cet.AO staining and Annexin V-PE/7-AAD double staining proved that combination with Cet and ORI synergistically induced LSCC cells apoptosis.Flow cytometry using propidium iodide(PI)staining assay showed that ORI plus Cet strongly caused G2/M phase arrest in two LSCC cell lines.The cell number of G2/M phase decreased after treatment with ORI/Cet for 12 h,while there was a significant increase in apoptotic cells,indicating that ORI/Cet-caused a shift from G2/M arrest to apoptosis.In vivo,the results of TUNEL assay demonstrated that combination treatment significantly increased the percentage of apoptotic cells compared with the cells of mono-treated mice.Extrinsic and intrinsic pathways are two major ways to induce apoptosis by some antitumor regents in tumor.In this study.we observed that Cet enhanced ORI-induced up-regulation of Fas and FasL and the activation of caspase 8.The results of High Content Screening and flow cytometry showed that combination treatment decreased mitochondrial membrane potential.In addition.Cet enhanced ORI-induced up-regulation of Bax and down-regulation of Bcl-2.These results demonstrated that both intrinsic and extrinsic apoptotic pathways were associated with apoptosis by combination of ORI and Cet in LSCC cells.Furthermore.western blotting showed that ORI plus Cet significantly increased the levels of endoplasmic reticulum(ER)stress related protein Grp78 and CHOP.suggesting that ORI/Cet induced apoptosis via the activation of ER stress.Reactive oxygen species(ROS)act as secondary messengers and are essential for many physiological processes,such as cell proliferation,cell cycle,differentiation,apoptosis,and so on.We also found that combination of ORI and Cet could rapidly induce ROS production in 3 hours,and subsequently decrease to normal levels,while the number of G2/M phase and apoptosis was increased.Further,a general free radical scavengers(NAC)reversed ORI/Cet-induced up-regulation of Fas,Bax,Grp78 and CHOP and the down-regulation of Bcl-2.Pretreatment with NAC inhibited G2/M arrest,indicating that ROS might be an early mediator to promote ORI/Cet induced LSCC cell apoptosis and G2/M arrest.In addition.ORI plus Cet increased the levels of ROS in HEp-2 xenograft tumor model.Therefore.ROS generation by combination of ORI and Cet promotes LSCC cells death and the inhibition of HEp-2 xenograft tumor growth.In the present study,we found that MAPK cascades were involved in LSCC cells death induced by ORI/Cet.We observed that combination of ORI and Cet significantly increased the phosphorylation of JNK in vivo and in vitro,an important subgroup of MAPK super family.Additionally,inhibition of JNK by SP600125 decreased.in part.ORI/Cet-induced apoptosis and G2/M arrest.Further,NAC significantly inhibited the up-regulation of p-JNK in LSCC cells.suggesting that ORI/Cet-induced JNK signaling was mediated through ROS,which induced LSCC cell death.In tumor cells,NF-κB,as a main transcription factor.was associated with apoptosis by various anticancer agents.We observed that combination of ORI with Cet significantly inhibited the expression of nuclear NF-κB through inhibiting the phosphorylation of IκB.NF-κB inhibitor PDTC or MG132 could increase ORI/Cet-caused apoptosis.suggesting that N F-κB protected the LSCC cell death induced by ORI/Cet.Further.the results from High Content Screening showed that combination treatment could inhibit TNF-α induced NF-κB nuclear translocation.We found that inhibition of NF-κB by PDTC further decreased the downregulation of Bcl-2.indicating that NF-κB-mediated dowmegulation of Bcl-2 is one of mechanism related with combination treatment-induced apoptosis.In addition,the inhibition of Akt and STAT further increased the downregulation of nuclear NF-κB.These results suggested that the downregulation of the expression of nuclear NF-κB through PI3K/Akt and JAK/STAT pathway and inhibition of NF-κB nuclear translocation were involved with ORI/Cet-induced LSCC cell death.In recent years,autophagy has gradually become a research field in molecular biology.Furthermore,we observed that combination of ORI and Cet caused autophagy vesicle and GFP-LC3 green fluorescence spots by fluorescence microscopy after MDC staining and GFP-LC3 plasmid transfection,suggesting that ORI/Cet induced autophagy in LSCC cell.Owing to the fact that combination of ORI and Cet caused both autophagy and apoptosis in LSCC cells,we further analyzed the mechanism of autophagy and the relation between apoptosis and autophagy.In these two LSCC cells with different p53 status,combination treatment upregulated p-AMPK while p-mTOR was downregulated.In addition.inhibiting AMPK/mTOR pathway reduced ORI/Cet-induced autophagy.indicating that activation of AMPK/mTOR signaling pathway promoted ORI/Cet induced autophagy in two LSCC cell lines,which is independent of p53 status.Additionally,we demonstrated that ORI/Cet induced the up-regulation of Beclin 1 protein expression and promoted NF-κB binding on the Becn 1 promoter,which was coupled to increased autophagy.Both SiRNA Beclin 1 and autophagy inhibitors(3-MA and chloroquine)could increase ORl/Cet induced-cell growth inhibition and apoptosis,indicating that autophagy induced by these two agents plays a cytoprotective role and anti-apoptotic effects.Notably,at 48 hours following the combined treatment,autophagy began to decrease but apoptosis was significantly elevated,suggesting that combined treatment might cause a switch from autophagy to apoptosis in long time term.In summary;Cet bind to the extracellular region of EGFR,while ORI bind to the EGFR intracellular region of EGFR.This research firstly reported combination treatment caused LSCC cells death through duel inhibition of EGFR.We demonstrated that combination of ORI and Cet induced G2/M phase arrest,apoptosis and autophagry and related mechanism through in vivo and in vitro experiments,which provided theoretical and practical basis for clinical application of laryngeal squamous cell carcinoma.