Synergistic Inhibition of Thalidomide and Icotinib on Human Non-Small Cell Lung Carcinomas Through ERK and AKT Signaling

Abstract

BACKGROUND Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used in the treatment of non-small cell lung cancer (NSCLC) patients with sensitive EGFR mutations. However, the survival of patients with EGFR-TKI administration is limited by the inevitable development of acquired drug resistance. Recently, multi-targeted drugs combination has been shown to be a promising strategy to improve the efficacy of EGFR-TKI treatment and enable the reduction of drug resistance in NSCLC. MATERIAL AND METHODS Humanized NSCLC cell lines PC9 and A549 were co-cultured with thalidomide and/or icotinib to test for anti-tumor efficiency. Cell proliferation was measured by MTT assay, cell apoptosis by flow cytometry and cell migration by wound healing assay. Western blot was performed to determine the expression of caspase-3, -8, -9, Bax, EGFR, VEGF-R, AKT, ERK, MMP2, MMP9, and NF-κB. The xenograft mouse model was used to explore the effects of thalidomide and icotinib in vivo. Immunohistochemical testing was used to determine the expression of Ki-67 and TUNEL staining in tumor tissues. RESULTS Treatments of thalidomide and/or icotinib reduced cell viability, induced apoptosis, and suppressed migration. Attenuation of pEGFR and pVEGF-R resulted in deactivation of ERK and AKT pathways, which eventually increased the anti-proliferative response. In PC9 xenograft model, combined administration of thalidomide and icotinib restrained tumor growth with remarkable reduced Ki-67 index and increased TUNEL positive cells. CONCLUSIONS Thalidomide sensitizes icotinib to increase apoptosis and prevent migration, and it may be a potentially promising anti-tumor drug in lung cancer multi-modality therapy.

Figure 1

Effects of thalidomide and icotinib on proliferation of PC9 and A549 cells. (A, B) Icotinib inhibited proliferation of PC9 and A549 cells increasingly with the elevation of drug concentration. PC9 and A549 cells were treated with various doses of icotinib (0, 0.1, 0.5, 1, 2.5, 5, 10, and 20 μM) for 24 hours. MTT assay was used to determine cell viability. (C, D) Combination treatment with thalidomide (50, 100, 200, and 400 μg/mL) and icotinib inhibited cell proliferation in PC9 and A549 cells. Data are represented as the means ±SD, n=3, * P<0.05, ** P<0.01. (E, F) Combination index and fractional effect values of thalidomide and icotinib combination treatment in PC9 and A549 cells.

Figure 2

Thalidomide and icotinib induced apoptosis in PC9 cells. (A, B) Flow cytometry analysis showed the percentage of apoptotic cells in the control group, thalidomide group, icotinib group, and combination group. (C, D) Western blot analysis showed the expression of caspase-3, -8, -9, and Bax in the control group, thalidomide group, icotinib group, and combination group. Data are represented as the means ±SD, n=3. * P<0.05, ** P<0.01. Relative rate of Bax was put on the left Y axis and caspase-3, -8, -9 on the right Y axis.

Figure 3

Thalidomide and icotinib inhibited migration of PC9 cells. (A) Images of wounded PC9 cells (200×). (B) Quantitative analysis of the percentage of open wound area during and after the 24 hours incubation. Data are represented as the mean ±SD, n=4, * P<0.05, ** P<0.01. (C, D) Western blot analysis showed the expression of MMP2, MMP9, NF-κB p65, and phospho-NF-κB p65 in the control group, thalidomide group, icotinib group, and combination group. β-actin was used as a loading control. Data are represented as the means ±SD, n=3, * P<0.05, ** P<0.01.

Figure 4

Thalidomide and icotinib inhibited the EGFR and VEGF-R2 pathways in PC9 cells. Western blot analysis showed the expression of pEGFR, pVEGF-R2, AKT, pAKT, ERK, and pERK in PC9 cells in the control group, thalidomide group, icotinib group, and combination group. β-actin was used as a loading control. Data are represented as the means ±SD, n=3, * P<0.05, ** P<0.01.

Figure 5

Thalidomide and icotinib inhibits tumor growth and promote tumor death in vivo. (A, B) PC9 tumor cells were injected subcutaneously. Mice were divided into groups as follow: control, thalidomide, icotinib, and combination group. Data represent the mean tumor volume of 5 mice. The tumor size in combination group is significantly smaller than that in individual icotinib group and individual thalidomide group. Data are represented as the means ±SD, n=5, * P<0.05, ** P<0.01. (C) Immunohistochemical reaction to Ki-67 and TUNEL staining in control, thalidomide, icotinib and combination group (200×).