Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

Abstract

Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and recorded, and at the end of experiments, tumor xenografts were removed for Western blot and immunohistochemical analyses. Compared to control groups (negative control, regular-dose icotinib [IcoR], high-dose icotinib [IcoH], and docetaxel [DTX]) and regular icotinib dose (60 mg/kg) with docetaxel, treatment of mice with a high-dose (1200 mg/kg) of icotinib plus sequential docetaxel for 3 weeks (IcoH-DTX) had an additive effect on suppression of tumor xenograft size and volume (P < 0.05). Icotinib-containing treatments markedly reduced phosphorylation of EGFR, mitogen activated protein kinase (MAPK), and protein kinase B (Akt), but only the high-dose icotinib-containing treatments showed an additive effect on CD34 inhibition (P < 0.05), an indication of reduced microvessel density in tumor xenografts. Moreover, high-dose icotinib plus docetaxel had a similar effect on mouse weight loss (a common way to measure adverse reactions in mice), compared to the other treatment combinations. The study indicate that the high dose of icotinib plus sequential docetaxel (IcoH-DTX) have an additive effect on suppressing the growth of wild-type EGFR NSCLC cell nude mouse xenografts, possibly through microvessel density reduction. Future clinical trials are needed to confirm the findings of this study.

Keywords: docetaxel; epidermal growth factor receptor (EGFR); icotinib; non-small cell lung cancer; nude mouse xenografts.

Figure 1. Effects of different treatments on modulation of tumor xenograft volume and size

The mice were subcutaneously injected with NSCLC A549 cells and when tumor xenografts reached 5–6 mm³, the mice received different treatments (n = 8 mice/group) for up to 6 weeks. (A) Comparison of single drug treatment with the negative control. During the treatment, tumor xenograft volumes were measured twice a week for 3 weeks. (B) Comparison of a regular icotinib dose plus or sequential docetaxel treatment with the negative control. C. Comparison of a high icotinib dose plus or sequential docetaxel treatment with the negative control. Control, negative control; DTX, docetaxel; IcoR, a regular icotinib dose; IcoH, a high icotinib dose; IcoH-D, a high icotinib dose and sequential docetaxel for 3 weeks; IcoR-D, a regular icotinib dose and sequential docetaxel for 3 weeks; IcoH+D, a high icotinib dose plus docetaxel for 3 weeks; IcoR+D, a regular icotinib dose plus docetaxel for 3 weeks; D-IcoR, docetaxel and sequential regular icotinib dose for 3 weeks; D-IcoH, docetaxel and sequential high icotinib dose for 3 weeks.

Figure 2. Expression and phosphorylation of tumor-related proteins in tumor xenografts

The mice were subcutaneously injected with NSCLC A549 cells and when tumor xenografts reached 5–6 mm³, the mice received different treatments for up to 6 weeks. After the experiments, tumor xenografts were resected and subjected to protein extraction and Western blot analysis of EGFR, MAPK, and Akt protein level and phosphorylation. C, negative control group; D, docetaxel; IcoR, a regular icotinib dose; IcoH, a high icotinib dose; IcoH-D, a high icotinib dose and sequential docetaxel for 3 weeks; IcoR-D, a regular icotinib dose and sequential docetaxel for 3 weeks; IcoH+D, a high icotinib dose plus docetaxel for 3 weeks; IcoR+D, a regular icotinib dose plus docetaxel for 3 weeks; D-IcoR, docetaxel and sequential regular icotinib dose for 3 weeks; D-IcoH, docetaxel and sequential high icotinib dose for 3 weeks.

Figure 3. Immunohistochemical detection of CD34 expression in tumor xenografts

The mice were subcutaneously injected with NSCLC A549 cells and when tumor xenografts reached 5–6 mm³, the mice were received different treatments for up to 6 weeks. At the end of experiments, tumor xenografts were resected and subjected to immunohistochemical analysis of CD34 level. Control, negative control; DTX, docetaxel; IcoR, a regular icotinib dose; IcoH, a high icotinib dose; IcoH-D, a high icotinib dose and sequential docetaxel for 3 weeks; IcoR-D, a regular icotinib dose and sequential docetaxel for 3 weeks; IcoH+D, a high icotinib dose plus docetaxel for 3 weeks; IcoR+D, a regular icotinib dose plus docetaxel for 3 weeks; D-IcoR, docetaxel and sequential regular icotinib dose for 3 weeks; D-IcoH, docetaxel and sequential high icotinib dose for 3 weeks.