Anti-tumor activity of erlotinib in the BxPC-3 pancreatic cancer cell line

Abstract

Aim: To investigate the effect and mechanism of action of erlotinib, an epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitor (TKI), in the human pancreatic cancer cell line BxPC-3 both in vitro and in vivo.

Methods: In vitro, human pancreatic cancer cell line BxPC-3 was exposed to varying concentrations of erlotinib, and its effects on proliferation, cell cycle distribution, apoptosis and the expression of pro- and antiapoptotic factors such as bcl-2, bcl-xl, bax and bak, and the expression of vascular endothelial cell growth factor (VEGF) were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis, terminal deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL), and reverse transcription-polymerase chain reaction (RT-PCR). Potential effect of erlotinib on angiogenesis was examined by tube formation assay. Tumor growth suppression was observed in xenografted nude mice with pancreatic cancer in vivo. Immunohistochemical (IHC) staining for EGFR and factor VIII-related antigen was undertaken to detect the microvessel density and VEGF expression in tumor tissue in xenograft nude mice.

Results: Erlotinib, as a single agent, repressed BxPC-3 cell growth in a dose-dependent manner, triggered G(1) arrest and induced cell apoptosis, and suppressed capillary formation of endothelium in vitro. Expressions of VEGF were significantly down-regulated at a high concentration of 200 mumol/L, however, the expressions of bcl-2 and bcl-xl were decreased at 50 mumol/L. In vivo, Erlotinib-treated mice demonstrated a reduced tumor volume, weight and microvessel density as compared to the control. IHC staining showed decreased expression of EGFR and RT-PCR had lower VEGF expression in treated mice.

Conclusion: The in vitro and in vivo findings provide evidence that BxPC-3 cells are inhibited with erlotinib treatment. Inhibition of EGFR may be a promising adjuvant chemotherapy strategy in pancreatic cancer treatment.

Figure 1

A: Growth percentage of BxPC-3 cell line after 72 h of exposure to erlotinib at varying concentrations ranging from 1-100 μmol/L. BxPC cell proliferation was significantly suppressed by erlotinib at different concentrations; B: Growth percentage of BxPC-3 cell line after exposure to erlotinib at a concentration of 50 μmol/L at different time points.

Figure 2

Induction of apoptosis in BxPC-3 cell line. There was a significant apoptosis in erlotinib treated group both in flow cytometry analysis and TUNEL assay after 48 h treatment (^aP < 0.05).

Figure 3

A: Effect of erlotinib on mean tumor volume in the BxPC-3 xenograft model. Mice were implanted with BxPC-3 cells. When palpable tumors were established, animals were randomly divided into two groups. The animals were continuously gavaged with the agents for 4 wk as described in Matericals and Methods. Tumor size was measured twice per week. Values are means, n = 6. B: Mice of control group; C: Mice of erlotinb group.

Figure 4

Effects of erlotinib on capillary formation by ECV304 were examined in vitro. A: When cultured on ECMatrix, ECV304 cells rapidly aligned with hollow tube-like structures; B: ECV304 cells were treated with erlotinib (100 μmol/L), and significant inhibition of tube formation was achieved compared with the control.

Figure 5

Expression of EGFR and the blood vessel endothelial cells in different treatment group in BxPC-3 mouse xenograft tissues. IHC was used to determine expression levels of EGFR and evaluate tumor microvessel density. A: HE staining for each sample (× 400); B: Expression of EGFR in treatment group was decreased compared with the control (× 400); C: Microvessel density of erlotinib treated group was lower than that of the control group.

Figure 6

Expressions of EGFR, Bcl-2, Bcl-xl, Bax, Bak and VEGF mRNA in BxPC-3 cells treated with different concentrations (μmol/L) of erlotinib for 48 h and xenograft tissues were detected by RT-PCR. A: Effects of erlotinib on the expressions of EGFR, apoptosis-associated factors and VEGF. Lane M: DNA marker; Lane C: Control; other lanes: Different concentrations of erlotinib. B: Effects of erlotinib (100 mg/kg daily) on expression of VEGF in tumor tissues. Lane M: DNA marker; Lane 1, 2: Control group; Lane 3, 4: Treatment group. RT-PCR for GAPDH was performed in parallel and showed an equal amount of total RNA in the sample.