Combination Effects Of Nimotuzumab With Cisplatin Or Fluorouracil On Human Esophageal Squamous Cell Carcinoma Cells In Vitro

BackgroundOverexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis. As reported, EGFR is over expressed in a variety of cancers including esophageal squamous cell carcinoma (ESCC), which is found to be upregulated in30-90%of ESCC. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has demonstrated encouraging clinical results in the absence of severe side-effects observed with other approved anti-EGFR antibodies. Most studies have mainly focused on head and neck squamous cell carcinomas (HNSCCs) and brain malignancies, not on ESCCs.ObjectiveTo investigate the inhibitory proliferation effect of h-R3combined with chemotherapeutic drugs on human ESCC cell lines with different EGFR expression levels and whether the responses are correlated with the level of EGFR expression, and to explore the possible molecular mechanism.Methods1. EGFR expression on EC1and EC9706cell lines was detected by immuno-histochemisty.2. The inhibitory effect of cisplatin (also known as DDP) and fluorouracil (also known as5-FU), used alone or in combination with h-R3, on the proliferation of EC1and EC9706cells in vitro was assessed by MTT assay. Cells were incubated with h-R3(12.5,25,50,100,200,400μg/ml) alone, DDP (0.25,0.5,1,1.5,2,2.5μg/ml) alone,5-FU (0.5,1,2,4,6,8μg/ml) alone. And30%inhibitory concentration (IC30) of DDP and5-Fu was gained to be used as the following experimental concentration, with h-R3(100μg/ml) plus IC30of DDP or5-Fu for48hours respectively. Treatment groups consisted of control, h-R3alone, DDP alone, DDP alone, the combination of h-R3and DDP, and the combination of h-R3and5-FU. The inhibitory effect of cells were detected by MTT assay after treatment with the inhibitory rates of cells calculated according to OD value by the following equation: Rate of growth inhibition (%)=(1-Atreated/Acontrol) x100%, and q value was calculated when the two drugs combined.3. Flow cytometry assay was applied to analyze cell cycle distribution after cells were collected48h after h-R3treatment (100μg/ml).4. Apoptosis was performed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay after different drugs alone or combination effect on the two cells.Results1. EGFR expression in human ESCC cell lines differs in EGFR status:our immunohistochemisty analysis demonstrated that EC9706cells express high levels of EGFR on the cytomembrane and cytoplasma91.22±4.65%, whereas EC1cells showed a low level of EGFR expression22.45±2.54%.2. H-R3treatment alone did not decrease EC1and EC9706cells viability dramatically, regardless of the concentration used. The single drug of h-R3had effect on EC1and EC9706cells, after48-hour treatment, and the highest proliferation inhibition rates were10.10±0.58%and27.84±2.42%, respectively (P<0.01). The single drug of DDP and5-FU both inhibited the growth of EC1and EC9706cells, and the IC30were0.5μg/ml for DDP and1μg/ml for5-FU, respectively. The combination of h-R3with DDP or5-FU in EC9706cells had higher proliferation inhibition rates than the effect of single agent (P<0.01), but the two drugs did not have synergistic role on the proliferation inhibition by q value (0.85<q<1.15). The combination of h-R3with DDP or5-FU in EC1cells synergistically did not have higher proliferation inhibition rates than the effect of single agent (P>0.05).3. An obvious shift into the G1phase of the cell cycle was revealed in EC9706cell lines after48hours of100μg/ml h-R3treatment, not in EC1cell lines.4. H-R3treatment alone induced slightly apoptosis of EC9706cells (P<0.05), but not in EC1(P>0.05). The single drug of DDP and5-FU both induced apoptosis of EC1and EC9706cells. The combination of h-R3with DDP or5-FU in EC9706cells had higher apoptosis rates than the effect of single agent (P<0.05), but the two drugs did not have synergistic role on the apoptosis (P<0.05). The combination of h-R3with DDP or5-FU in EC1cells did not have higher apoptosis rates than the effect of single agent (P>0.05).Conclusions1. The inhibitory effects of h-R3may have relation to the expression of EGFR.2. H-R3alone could slightly inhibit the growth of EC9706, and induced an obvious shift into the G1phase of the cell cycle and had cooperative growth inhibitory with chemotherapy (DDP and5-FU), but two drugs did not have synergistic role.3. Similar results were not found in EC1when treated with h-R3alone or plus chemotherapy.