Enhancing chemosensitivity in oral squamous cell carcinoma by lentivirus vector-mediated RNA interference targeting EGFR and MRP2

Abstract

Oral cancer is the eighth most common type of cancer among men worldwide, with an age-standardized rate of 6.3 per 100,000, and is the fourth leading cause of cancer-associated mortality among men in Taiwan. Cisplatin and 5-fluorouracil (5-FU) are two of the most frequently utilized chemotherapy drugs for the treatment of oral cancer. Although oral cancer patients initially benefit from chemotherapy with these drugs, they may develop resistance to them, which worsens their prognosis and reduces survival rates. It has been reported that increased levels of epidermal growth factor receptor (EGFR) and multidrug resistance-associated protein 2 (MRP2) induce drug resistance in numerous types of human cancer. Therefore, the present study employed lentivirus vector-mediated RNA interference (RNAi) in order to target the genes encoding EGFR and MRP2 in the oral squamous cell carcinoma cell line OC2. It was observed that RNAi-mediated downregulation of EGFR or MRP2 increased the sensitivity to 5-FU and cisplatin in OC2 cells. Downregulation of EGFR resulted in significant suppression of OC2 tumor growth following 5-FU administration. However, simultaneous downregulation of the two genes did not further suppress the tumor growth, indicating that MRP2 does not have a significant role in the chemosensitivity of EGFR-downregulated cells to 5-FU. In contrast, downregulation of MRP2 was demonstrated to significantly enhance the therapeutic effects of cisplatin in EGFR-downregulated OC2 tumors. The observation that the expression of MRP2 was positively correlated with the level of cisplatin resistance in cells suggests that RNAi-mediated downregulation of MRP2 may be applicable as a therapeutic approach toward reversing MRP2-dependent cisplatin resistance in oral cancer.

Keywords: RNA interference; chemosensitivity; epidermal growth factor receptor; multidrug resistance-associated protein 2; oral cancer.

Figure 1.

Relative expression levels of EGFR and MRP2 in OSCC cell lines. The messenger RNA levels of EGFR and MRP2 in OSCC cell lines were quantified using reverse transcription-polymerase chain reaction. The expression levels of EGFR and MRP2 in OC2 cells were assigned a value of 100% as controls. Data are presented as the mean ± standard error. *P<0.05. EGFR, epidermal growth factor receptor; MRP2, multidrug resistance-associated protein 2; OSCC, oral squamous cell carcinoma.

Figure 2.

Downregulation of EGFR expression inhibits the growth of OC2 cells. (A) Western blot analysis of EGFR protein (170 kDa) expression in OC2 cells transfected with shEGFR or shRFP. β-actin (42 kDa) was used as a loading control. (B) The effect of EGFR downregulation on the proliferation of OC2 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. The increase in cell populations was compared with the number of cells on day 0, which was assigned a value of 1. A significant difference was observed between the proliferation of shEGFR- and shRFP-transfected OC2 cells. (C) Quantitative analysis of the number of colonies formed using the colony formation assay. A significant difference was observed between the colony-forming ability of shEGFR- and shRFP-transfected OC2 cells. Data are presented as the mean ± standard error. *P<0.01. EGFR, epidermal growth factor receptor; sh, small hairpin; RFP, red fluorescent protein.

Figure 3.

Downregulation of EGFR and MRP2 enhances the sensitivity of OC2 cells to 5-FU. (A) Reduced expression of EGFR and MRP2 in OC2/shEGFR/shMRP2 cells. The messenger RNA levels of EGFR and MRP2 in the cells were quantified using reverse transcription-polymerase chain reaction analysis. The expression levels of EGFR and MRP2 in OC2/shRFP were assigned a value of 100%. (B) Determination of the IC₅₀ values of 5-FU in shRNA-transfected OC2 cells. The IC₅₀ value of 5-FU in OC2/shRFP was used as the control. (C) Diagram illustrating the procedure of the in vivo experiments. A total of 5×10⁶ shRNA-transfected OC2 cells were subcutaneously implanted into the right dorsal flank of nude mice (n=6/group). When the tumor volumes reached ~100 mm³ (set as day 0), the mice were administered intraperitoneal injections of 5-FU (15 mg/kg) for a total of 6 treatments. The tumor volumes were measured daily. Solid arrow indicates 5-FU treatments on days 0, 1, 2, 7, 8 and 9. (D) In vivo therapeutic effect achieved by the downregulation of EGFR and MRP2. The tumor volumes on day 0 were assigned a value of 100%. The tumor volumes of OC2/shRFP group were used as the controls. Data are presented as the mean ± standard error. *P<0.05. **P<0.01. EGFR, epidermal growth factor receptor; MRP2, multidrug resistance-associated protein 2; 5-FU, 5-fluorouracil; sh, small hairpin; RFP, red fluorescent protein; IC₅₀, half-maximal inhibitory concentration; s.c., subcutaneously; i.p., intraperitoneal.

Figure 4.

Development of the OC2CisR cell line. (A) Expression of EGFR and MRP2 in OC2CisR cells. The messenger RNA levels of EGFR and MRP2 in OC2CisR cells were quantified using reverse transcription-polymerase chain reaction analysis. The expression levels of EGFR and MRP2 in OC2 cells were assigned a value of 100% as controls. (B) Determination of the IC₅₀ values of cisplatin in cells. The IC₅₀ value of cisplatin in OC2 was used as the control. (C) Cell apoptosis assay. OC2 and OC2CisR cells were exposed to increasing concentrations of cisplatin ranging from 1 to 16 µg/ml for 24 h, and stained with Annexin V-fluorescein isothiocyanate and propidium iodide, followed by flow cytometric analysis. The percentage of total apoptotic cells is shown. (D) Diagram illustrating the procedure of the in vivo experiments. A total of 8×10⁶ cells were subcutaneously implanted into the right dorsal flank of nude mice (n=6/group). When the tumor volumes reached ~100 mm³ (set as day 0), the mice were administered intraperitoneal. injections of cisplatin (3 mg/kg) for a total of 3 treatments. The tumor volumes were measured daily. Solid arrow indicates cisplatin treatment on days 0, 7 and 14. (E) Comparison of tumor growth rates in the mice following cisplatin administration. The tumor volumes on day 0 were assigned a value of 100%. The interaction of time and group in the generalized linear model with generalized estimated equation was 52.1% (P=0.024), which indicated that the linear growth rate was 52.1% higher per day for the OC2CisR group compared with the OC2 group. Data are presented as the mean ± standard error. *P<0.05. CisR, cisplatin-resistant; EGFR, epidermal growth factor receptor; MRP2, multidrug resistance-associated protein 2; IC₅₀, half-maximal inhibitory concentration; s.c., subcutaneously; i.p., intraperitoneal.

Figure 5.

Downregulation of EGFR and MRP2 increases the inhibitory effects of cisplatin on the growth of OC2CisR tumors. (A) Reduced expression of EGFR and MRP2 in OC2cisR/shEGFR/shMRP2 cells. The messenger RNA levels of EGFR and MRP2 in cells was quantified using reverse transcription-polymerase chain reaction analysis. The expression levels of EGFR and MRP2 in OC2/shRFP were assigned a value of 100% as controls. (B) Therapeutic effect achieved by the downregulation of EGFR and MRP2 in OC2CisR tumors. The procedure of the in vivo experiments is illustrated in Fig. 4D (n=6/group). The tumor volumes on day 0 were assigned a value of 100%. The interaction of time and group in the generalized linear model with generalized estimated equation was 37.4% (P=0.035), which indicated that the linear growth rate was 37.4% higher per day for the OC2CisR/shEGFR group compared with the OC2CisR/shEGFR/shMRP2 group. Data are presented as the mean ± standard error. *P<0.05. EGFR, epidermal growth factor receptor; MRP2, multidrug resistance-associated protein 2; CisR, cisplatin-resistant; sh, small hairpin; RFP, red fluorescent protein.