Effects Of Cetuximab In Human Nasopharyngeal Carcinoma Cell Lines

Background and ObjectiveNasopharyngeal carcinoma (NPC) in China is characterized as poor differentiation and prone to metastasis. Cisplatin (DDP) based chemotherapy is an integral part of treatment for patients with local advanced or recurrent and metastatic nasopharyngeal carcinoma. Concurrent chemoradiotherapy has significantly improved the cure rate of local advanced cases than radiotherapy alone. Palliative chemotherapy can prolong the survival time in the metastatic setting. However, the DDP based regimen exhibits higher toxicity, and the extent of delivering high doses or high intensified DDP has been largely limited. Moreover, most advanced NPC patients eventually die of developing primary or secondary multi-drug resistance and further metastases. All of these urge for more effective therapies, and the combination of biologic targeted agents with conventional modalities is one of the promising strategies investigated to further improve the therapeutic effect of advanced NPC nowadays. Cetuximab (Erbitux, C225), a monoclonal antibody specifically targeted at the epidermal growth factor receptor (EGFR) which usually over expressed on the cell membrane of most epidermoid malignancy including NPC, has been demonstrated great radiosensitizing activity and synergistic effect when combined with radiotherapy in the treatment of local advanced head and neck cancer. However, it remains controversy about the combination effect of C225 and DDP in platinum refractory advanced NPC therapy. The present study was designed to evaluate the effect of C225 alone or in different sequence of combination with DDP in human nasopharyngeal carcinoma parental cell line HNE1 and cisplatin-resistant cell line HNE1/CDDP, and to investigate the effect of C225 alone on the metastatic ability of both NPC cell lines and the tubule formation of human umbilical vein endothelial cells in growth factor reduced matrigel (GFR- matrigel).MethodsHuman nasopharyngeal carcinoma parental cell line HNE1 and its cisplatin resistant cell line HNE1/CDDP and human umbilical vein endothelial cell line CRL-2480 (HUVEC-12) were used as in vitro models. In part one, immuocytochemistry was used to determine EGFR expression in both NPC cell lines. The growth inhibition effect of C225 alone or in combination with DDP in both NPC cells was determined by Methyl thiazolyl tetrazolium (MTT) reduction assay or colony formation assay. And the combination effect of C225 and DDP was analyzed. In part two, In vitro attachment, migration and invasion of both NPC cells on matrigel were detected by MTT, wound healing and Transwell assays respectively. EGFR expression in CRL-2480 cells was also examined by immuocytochemistry. Tubule formation assay in GFR-matrigel was carried out to determine anti-angiogenesis effect of C225 in CRL- 2480 cells.ResultsPart one:1 EGFR was over expressed in HNE1 and HNE1/CDDP cells.2 At the concentration of 1 to 200μg/ml, the growth inhibition effect of C225 alone in HNE1 and HNE1/CDDP cells was dose independent, and the maximum growth inhibition rates of C225 in HNE1 and HNE1/CDDP cells were (28.17±3.47)% and (17.65±1.73)%, respectively. The inhibition effect was greater in HNE1 than in HNE1/CDDP.3 The combination effect of C225 (10μg/ml or 80μg/ml) with DDP at concentration approximately or lower than IC50 in both NPC cells was additive and independent on the sequence of drug administration. The combination effect of C225 with DDP at concentration further higher than IC50 was less than additive.Part two:1 C225 alone could significantly attenuate the adherent abilities in HNE1 and HNE1/CDDP cells. The number of cell adhesion on matrigel in the C225 treatment group was less than the control group in both cell lines (both P value<0.05).2 C225 significantly reduced the number of invasive cells in HNE1 and HNE1/CDDP cells. The invasive cell number in the C225 treatment group was less than the control group in both NPC cell lines (both P value<0.01).3 C225 significantly reduced the migration distance in HNE1 and HNE1/CDDP cells. Cell migration distance in the C225 treatment group was shorter than the control group in both NPC cell lines (both P value<0.05).4 EGFR was over expressed in CRL-2480 cells.5 C225 could significantly shorten the length of tubules formed by CRL-2480 cells directly under the condition of lower growth factors, which was 341±15μm in the experimental group compared with 390±15μm in the control group (t=5.567, P =0.000). The indirect effect of C225 through down regulating HNE1 secretion on the tubule formation was not significantly (t=1.602, P =0.14).Conclusions1 EGFR was over expressed in HNE1, HNE1/CDDP and CRL-2480 cell lines.2 The effect of C225 alone was mild on inhibiting proliferation in HNE1 and HNE1/CDDP cells. 3 The combination effect of C225 with DDP at concentration approximately or lower than IC50 in both NPC cells was additive and independent on the sequence of drug administration. C225 could not overcome the resistance to cisplatin in HNE1/CDDP cells.4 C225 alone could attenuate the metastatic abilities of both NPC cells.5 C225 could shorten the length of tubule formed by CRL-2480 cells directly under the condition of lower growth factors. And the indirect effect of C225 through regulating HNE1 secretion on the tubule formation was not significantly.