| IntroductionHead and neck cancer is the seventh most common cancer worldwide with 90% cases being squamous cell carcinomas(HNSCC).Surgery and radiotherapy are the corner stones in the treatment of HNSCC.In recent years,concurrent chemoradiotherapy has gradually been established,the overall survival and the locoregional control of patients with locoregionally advanced HNSCC have been improved. However,there are still some problems in treatment regimes,such as the increased cytotoxic effects,the treatment resistance,the high recurrence rate and the low survival rate of 5 years.Thus,effectiveness of the treatment regime and predictions of the curative effect remain difficult questions for HNSCC.The cancer target therapy has some advantages with specificities to the tumor cells, strong anticancer effects and minimal toxicities to normal tissues.Targeted anticancer therapy is a biological therapy.It is based on a procedure that may cause the transformation of normal cells into cancer cells.It interferes with this procedure on a molecular level,thereby inhibiting tumor growth.One of the most promising strategies is the targeted anticancer therapy based on the epidermal growth factor receptor (EGFR).EGFR is ErbB receptor of the tyrosine kinase.The ErbB family contains four members:EGFR/ErbB1,HER2/ErbB2,HER3/ErbB3 and HER4/ ErbB4.Specific ligand binds to separate receptors by formation of active dimmers.This procedure is followed by auto-phosphorylation,activates a series of complex cellular signal pathways,regulates biological function of cells,such as cell proliferation,apoptosis, angiogenesis,cell adhesion and can lead to the development of cancer in abnormal events,further to invasiveness and metastasis.EGFR is overexpression in 80%-90%of SCC.Overexpression of the EGFR is correlated with a poor prognosis,a high recurrence rate,a low survival rate and resistance to chemotherapy and radiotherapy.Two predominant classes of EGFR inhibitors have been developed.On the one hand,monoclonal antibodies(mAbs),such as Cetuximab(Erbitux,IMC-C225),which is an IgG1 monoclonal antibody,blocks phosphorylation and activation of EGFR by binding with high affinity to the extracellular domain of EGFR.On the other hand, small molecule tyrosine kinase inhibitors(TKIs),such as Gefitinib(Iressa,ZD1839), which inhibits the intracellular kinase domain of EGFR.The function of targeting agent is only put tumor in the control,which can not kill the tumor.The rate of effectiveness of a majority of molecular targeting agents is approximately 10%.Exploring a targeting agent combined with chemo-radiotherapy is a treatment entailing less side effects than a traditional chemo-radiotherapy,making it a worthwhile object of study in the development of a cancer treatment.Some studies using cetuximab have shown enhanced efficacy in treatment of locoregionally advanced HNSCC when combined to radiotherapy or chemotherapy at present.Whether gefitinib has similar efficacy as part of radiotherapy is currently less well characterized.There is only little information on the potential enhancing cytotoxic effect when combining cetuximab with cisplatin-based chemoirradiation and predicting effective treatment in HNSCC.Using 10 established cell lines,we examined the effects of cetuximab and gefitinib as single agent and in a combined treatment with radiotherapy and subsequently compared them with one another.We examined the effects of ombining cetuximab with cisplatin-based chemoirradiation.The present study was performed to test the expression of the ErbB family members,and analyze prediction factors for curative effects to the EGFR inhibitor as a single agent and combined-modality paradigm on HNSCC in vitro.We hope to provide a new paradigm to further improve the therapeutic ratio in the treatment of HNSCC.Methods1.Cell linesTen recently established HNSCC cell lines were used,established in the University of Turku as described UT-SCC(Abbreviate as SCC).SCC-8,SCC-9, SCC-11,SCC-19a,SCC-29,SCC-34 and SCC-38 are SCCs of laryngeal origin (SCC-9 from a neck metastasis and SCC-11 from recurrence).SCC-24a1,SCC-24a2 and SCC-40 are SCCs from tongue.2.Methods(1) Cell culture:The cells were maintained in passage 15-30 by passing weekly or bioweekly in DMEM medium(10%FBS).Cells in mid-logarithmic growth were incubated at +37°C,were attached to the wall of the culture flask,and were used for the experiments.(2) Clonogenic growth assay:The cells were harvested with trypsin-EDTA, counted,and suspended in Ham's F-12 medium containing 15%FBS.The number of cells plated per well was adjusted to the plating efficiency(PE) of the cell line.The cells were incubated at +37°C for 4 weeks after indicated interference factors, whereafter the positive number of wells containing coherent,living colonies,consisting of 32 cells or more,were counted using an inverted phase-contrast microscope.(3) For protein expression and phosphorylation analysis of ErbB family member and key marks of EGFR signal pathway by Western blotting:cells were lysed and sample of total protein was separated on SDS-PAGE gels.Bound secondary antibody was visualized by ECL.(4) For mRNA expression of ErbB receptors by RT-qPCR:①The primers and Taqman probes designed using Primer Express software②Total RNA was extracted from cultured cells using Trizol③Construction of recombinant pEBS7- EGFR as a criterion,β-actin as an internal control.3.Experimental design(1) The IC70 and IC50 of drug(Cetuximab,Gefitinib,Cisplatin) sensitivity was determined using the 96-well plate clonogenic assay based on limiting dilutions.Each analysis with one cell line was divided into a negative control group and drug groups with different concentration.(2) The 96-well plate clonogenic assay was used to examine cetuximab or gefitinib combined with radiation on HNSCC in vitro.For example:cetuximab or gefitinib + radiation a= radiation group(fraction dose:0 Gy,0.75 Gy,1.25 Gy,2.5 Gy,5.0 Gy and 7.5 Gy)b= radiation + drug group(same radiation dose with above mention,IC70 of cetuximab or gefitinib)c= radiation + drug group(same radiation dose with above mention,IC50 of cetuximab or gefitinib)(3) The 96-well plate clonogenic assay was used to examine cetuximab,cisplatin combined with radiation on HNSCC in vitro.For example:cetuximab + cisplatin + radiationa= radiation group(fraction dose:0 Gy,0.75 Gy,1.25 Gy,2.5 Gy,5.0 Gy and 7.5 Gy)b= radiation + chemotherapy group(same radiation dose with above mention, cisplatin IC70)c= radiation + chemotherapy + drug group(same radiation dose with above mention,cisplatin IC70 and cetuximab IC70)d= radiation + chemotherapy + drug group(same radiation dose with above mention,cisplatin IC70 and cetuximab IC50)4.For evaluation of curative effects and data analysis(1) The PE was calculated using the formula -In(number of negative wells / total number of wells) / number of cells plated per well.(2) The drug(cetuximab,gefitinib,cisplatin) sensitivity was determined by the IC70 and IC50 values of each cell line,resulting in 30%and 50%inhibition in clonogenic survival.(3) The evaluation of curative effects to combined-modality paradigm①Fraction survival data as a function of the radiation dose with or without the indicated cetuximab and gefitinib doses were found to fit in the linear quadratic equation.A microcomputer program was used to fit data to F=exp[-(αD+βD2)].②The area under the curve(AUC) value,equivalent to mean inactivation dose, was obtained by numerical integration.The AUC-ratio(AUC for cetuximab or gefitinib + radiation +/- chemotherapy /AUC for radiation) and surviving fraction after the indicated doses of drugs were used to compare the effect of the drugs together with irradiation with the effect of irradiation alone.③Statistical comparisons were made using Student's t-test.The influence of the drug concentrations on the amount of supra-additive effect was tested by 1-way analysis of variance(ANOVA).The type of interaction was described by the terms.A additive,corresponding to the effect being equal with the calculated effects of the drug and radiation +/- chemotherapy.A supra-additive effect would indicate more than the effect of the additive.(4) For associations between ErbB expression and sensitivity to gefitinib, cetuximab and radiation were analyzed using Spearman correlation coefficient.Results1.For evaluation of curative effects to the EGFR inhibitor as a single agent and combined-modality paradigm on HNSCC in vitro(1) The drug sensitivity of 10 HNSCC cell lines①The IC70 concentration of cetuximab,gefitinib,cisplatin vary between 0.22 and 4.08 nM;0.036 and 4.8μM;0.038 and 0.22μg/ml.②The IC50 concentration of cetuximab,gefltinib,cisplatin vary between 0.58 and 8.2 nM;0.15 and 8.4μM;0.054 and 0.31μg/ml.③SCC-24a2 and SCC-40 were the most sensitive cell lines to cetuximab in the 10 HNSCC cell lines.SCC-24a2 and SCC-19a were the most sensitive cell lines to gefitinib in the 10 HNSCC cell lines.SCC-8 and SCC-34 were the most sensitve cell lines for cisplatin in 7 HNSCC cell lines.(2) The evaluation of curative effects to combined-modality paradigm of 7 HNSCC cell lines①Cetuximab combined with radiationThe effect of cetuximab in combination with radiation was additive(The effect of SCC-24a2 with IC50 concentration was additive,P=0.054),which is superior to radiation alone.The effect of SCC-24a2 with IC50 concentration was supra-additive (P＜0.05).②Gefitinib combined with radiation The effect of gefitinib in combination with radiation was additive,which is superior to radiation alone,except for SCC- 9,SCC- 24A2,SCC- 29,SCC- 34,and SCC- 40 with IC50 concentration,which were supra-additive(P＜0.05).③Cetuximab,cisplatin combined with radiationThe effect of simultaneous cetuximab,cisplatin combined with irradiation was at least additive,which is superior to radiation alone.SCC-40 and SCC-24a presented a supra-additive fashion in the concomitant exposure with cisplatin and both cetuximab concentrations.The two cell lines that reacted in a supra-additive manner to cetuximab combined with chemoirradiation were among the most sensitive to treatment with cetuximab as a single agent.2.For prediction of curative effects to the EGFR inhibitor as a single agent and combined-modality paradigm by testing key marks of EGFR signal pathway on HNSCC in vitro(1) For protein expression and phosphorylation analysis by Western blotting:Most HNSCC lines expressed variable amounts of EGFR,ErbB2,ErbB3 and pEGFR, pErbB2,pErbB3,pAkt,perk,whereas no signal was detected for ErbB4 in any of the 10 cell lines.(2) For ErbB mRNA expression by RT-qPCR:At mRNA level,the SCC cells expressed mostly EGFR followed by ErbB2,ErbB3 and ErbB4.(3) Association of ErbB expression with sensitivity to gefitinib:The association between high ErbB3 expression and high IC50 for gefitinib reached statistical significance(P = 0.02).The association between pErbB2 levels and IC50 for gefitinib was significant(P = 0.02).(4) Association of ErbB expression with sensitivity to cetuximab:EGFR protein expression reached a significant level of association with sensitivity to cetuximab(P = 0.048).(5) The cellular expression of phosphorylated ErbB3 was associated with resistance to radiation measured as AUC(P = 0.0045). Conclusions1.Concomitant use of cetuximab or gefitinib with radiation is superior to radiation alone and the gefitinib was superior to cetuximab in down-regulating HNSCC cell clonogenic growth when combined with radiation in HNSCC in vitro.The two cell lines in the most sensitive to cetuximab with IC50 concentration combined with radiation were the most sensitive to treatment with cetuximab as a single agent. Cetuximab was combined with chemo-radiotherapy,in which merely a low dose of cisplatin and cetuximab were used in order to achieve a significant curative effect in HNSCC in vitro.The effect of simultaneous cetuximab,cisplatin combined with irradiation was at least additive,which is superior to radiation alone.The two cell lines in a supra-additive manner to cetuximab combined with chemoirradiation were the most sensitive to treatment with cetuximab as a single agent.2.The pErbB2/ErbB3 protein expression reached a significant level of association with resistance to gefitinib.EGFR protein expression reached a significant level of association with sensitivity to cetuximab.The cellular expression of pErbB3 was associated with resistance to irradiation.Increased pErbB2/ErbB3 signaling may predict resistance to gefitinib,EGFR protein expression may predict sensitivity to cetuximab,and pErbB3 protein expression may predict resistance to irradiation in HNSCC.
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