Small interfering RNA targeting epidermal growth factor receptor enhances chemosensitivity to cisplatin, 5-fluorouracil and docetaxel in head and neck squamous cell carcinoma

Abstract

Overexpression of epidermal growth factor receptor (EGFR) has been found in various epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC), and is associated with increased tumor growth, metastasis, resistance to chemotherapeutic agents and poor prognosis. As such, EGFR is a potential target for antitumor therapy and several EGFR inhibitors have been investigated in preclinical or clinical settings. In the present study, we used small interfering RNA (siRNA) to downregulate EGFR expression while evaluating the effect of EGFR siRNA on cell proliferation, and the combined effects with cisplatin, 5-fluorouracil (5-FU) and docetaxel in HNSCC. Furthermore, HNSCC xenografts were treated with EGFR siRNA alone or in combination with cisplatin, and tumor growth was examined. EGFR expression, proliferation, angiogenesis and apoptosis index were evaluated by immunohistochemistry. The results showed that EGFR siRNA efficiently downregulated EGFR expression and inhibited cell growth of HNSCC. Treatment with EGFR siRNA in combination with cisplatin, 5-FU and docetaxel enhanced chemosensitivity with a significant increase in apoptosis. EGFR siRNA delivered by atelocollagen enhanced the antitumor effect of cisplatin in the HNSCC xenograft model. These cumulative results suggest that EGFR siRNA combined with cisplatin, 5-FU and docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with HNSCC.

Figure 1

Effects of epidermal growth factor receptor (EGFR) small interfering RNA (siRNA) on the expression of EGFR in HSC‐2 and SAS cells. (a) EGFR is overexpressed in HSC‐2 and SAS cells. Cell surface EGFR expression in HSC‐2 and SAS cells was determined by flow cytometry after staining with phycoerythrin (PE)‐conjugated antihuman EGFR monoclonal antibody (B4G7). Filled histograms represented non‐specific staining with mouse Ig‐PE (negative control). (b) Scrambled control siRNA did not significantly affect the expression of EGFR compared to mock transfected cells, whereas EGFR siRNA induced marked downregulation of EGFR expression. Bold line histograms represent the proportion of cells that were transfected with control (upper panel) or EGFR siRNA (lower panel). Filled histograms represent the proportion of mock transfected cells that were treated with TransIT‐TKO, but no siRNA (positive control). (c) EGFR expression was quantified by flow cytometry. The results were expressed as the percentage of intensity of fluorescence relative to controls (*P < 0.05). (d) HER2 expression was unaffected by either siRNA treatment. Filled histograms represented non‐specific staining with the secondary antibody without prior staining with the primary antibody (negative control). Thin line histograms represent the proportion of cells that were treated with TransIT‐TKO, but no siRNA (positive control). Dashed or bold line histograms represent the proportion of cells that were treated with control siRNA or EGFR siRNA, respectively. FITC, fluorescein‐isothiocyanate.

Figure 2

Epidermal growth factor receptor (EGFR) small interfering RNA (siRNA) suppressed the expression of EGFR and inhibited cell growth in HSC‐2 and SAS cells. (a) HSC‐2 and SAS cells were transfected with EGFR siRNA (50 nM), and scrambled siRNA‐transfected or mock transfected cells were used as controls. Mock transfected cells were treated with TransIT‐TKO, but no siRNA. The cells were harvested at the indicated time points and flow cytometric analysis was carried out. Upper panels show the inhibitory effects of EGFR siRNA on EGFR expression levels compared with mock transfected cells by flow cytometric analysis for 8 days post transfection. The lower panels show histograms representing the proportion of cells that were treated with EGFR siRNA (bold line) and mock transfected cells (filled) on day 3 post transfection. (b) HSC‐2 and SAS cells were transfected with 50 nM EGFR siRNA, control siRNA or mock transfected. At the indicated time points, the cells were trypsinized and counted by the trypan blue dye exclusion method in triplicate samples. The results represent the mean ± SEM. The experiment was repeated two additional times with similar results. ▪, mock transfected; ▿, control siRNA (50 nM); •, EGFR siRNA (50 nM). *P < 0.05 in comparison to the control siRNA. PE, phycoerythrin.

Figure 3

(a) Effects of epidermal growth factor receptor (EGFR) small interfering RNA (siRNA) on cisplatin, 5‐fluorouracil (5‐FU) and docetaxel sensitivity in HSC‐2 and SAS cells. In vitro cytotoxicity assays were carried out by MTS assays. Twenty‐four hours after transfection with EGFR siRNA, control siRNA (50 nM) or mock transfection cells were harvested and transferred to 96‐well plates. After treatment with cisplatin, 5‐FU or docetaxel for 48 h, MTS assay was carried out to determine the IC₅₀. The IC₅₀ is the concentration of cisplatin (left), 5‐FU (middle) or docetaxel (right) that caused a 50% reduction of absorbance relative to untreated cells. (b) Growth‐inhibitory effects of treatment with EGFR siRNA in combination with cisplatin, 5‐FU or docetaxel in HSC‐2 and SAS cells. HSC‐2 and SAS cells were treated with 50 nM control or EGFR siRNA for 24 h and were then exposed to cisplatin, 5‐FU or docetaxel (IC₅₀) for an additional 48 h. The results represent the mean ± SEM of three different experiments, and each was carried out in triplicate. *P < 0.05 in comparison to control siRNA. CDDP, cisplatin; DOC, docetaxel.

Figure 4

Effects of epidermal growth factor receptor (EGFR) small interfering RNA (siRNA) in combination with cisplatin, 5‐fluorouracil (5‐FU) or docetaxel on apoptosis in HSC‐2 (a) and SAS (b) cells. HSC‐2 and SAS cells were transfected with 50 nM control siRNA or EGFR siRNA for 24 h and were then exposed to cisplatin, 5‐FU or docetaxel (IC₅₀) for an additional 48 h. The IC₅₀ is the concentration that caused a 50% reduction of absorbance relative to untreated cells. Apoptotic cells were present in the area indicated by a bar on the left side of each histogram. The number in each panel represents the percentage of apoptotic cells as calculated by a flow cytometric analysis. The data represent one of three different experiments showing similar results. CDDP, cisplatin; DOC, docetaxel.

Figure 5

An in vivo study of epidermal growth factor receptor (EGFR) small interfering RNA (siRNA) in combination with cisplatin treatment in SAS human tumor xenografts. The mice were injected subcutaneously into the right flank with 3 × 10⁶ SAS cancer cells. When the tumors reached a volume of approximately 100 mm³, the mice were treated as follows: □, no treatment; ▵, atelocollagen; ○ and •, control and EGFR siRNA (5 µM/50 µL/dose intratumoral injection); ▿, cisplatin (1 µg/g/dose i.p); ▪ and ▴, combination of control or EGFR siRNA (5 µM/50 µL/dose intratumoral injection) and cisplatin (1 µg/g/dose i.p.), at the indicated time point. EGFR and control siRNA were mixed with atelocollagen. The tumor size was measured at the indicated time points and then the tumor volumes were calculated. Each group consisted of six mice. The values represented as mean ± SEM. EGFR siRNA‐treated mice versus cisplatin‐treated mice (no significant difference); EGFR siRNA plus cisplatin‐treated mice versus the cisplatin‐treated mice (*P = 0.0002) or control siRNA plus cisplatin (*P = 0.0027); control siRNA‐treated mice versus no treatment mice (no significant difference); control siRNA plus cisplatin‐treated mice versus cisplatin‐treated mice (no significant difference).

Figure 6

Effect of epidermal growth factor receptor (EGFR) small interfering RNA (siRNA) in combination with cisplatin on proliferation, angiogenesis and apoptosis in vivo. (a) Immunohistochemical analysis of SAS tumor xenografts (day 21) stained with hematoxylin and eosin (H&E), anti‐EGFR, anti‐Ki‐67 nuclear antigen, anti‐CD34 vessel staining and apoptosis by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate brotin nick‐end labeling (TUNEL) (magnification, ×400). (b) The quantification of proliferation index (Ki‐67), the microvessel density and the apoptosis index (TUNEL). All quantitative data are presented as mean ± SEM. *P < 0.05 in comparison to cisplatin alone.