Icotinib derivatives as tyrosine kinase inhibitors with anti-esophageal squamous carcinoma activity

Abstract

Previous report showed that a variety of icotinib derivatives bearing different 1,2,3-triazole moieties, which could be readily prepared via copper (I)-catalyzed cycloaddition (CuAAC) reaction between icotinib and different azides, exhibited interesting activity against different lung cancer cell lines such as H460, H1975, H1299, A549 or PC-9. To further expand the application scope of the compounds and to validate the function of triazole groups in drug design, the anti-cancer activity of these compounds against esophageal squamous carcinoma (ESCC) cells was tested herein. Preliminary MTT experiments suggested that these compounds were active against different ESCC cell lines such as KYSE70, KYSE410, or KYSE450 as well as their drug-resistant ones. Especially, compound 3l showed interesting anticancer activity against these cell lines. The mode of action was studied via molecular docking, SPR experiments and other biochemical studies, and 3l exhibited higher binding potential to wild-type EGFR than icotinib did. In vivo anticancer study showed that 3l could inhibit tumor growth of cell-line-derived xenografts in ESCC. Study also suggested that 3l was a potent inhibitor for EGFR-TK pathway. Combining these results, 3l represents a promising lead compound for the design of anti-cancer drugs against ESCC.

Keywords: 1,2,3-triazole; EGFR-TK pathway; ESCC; NSCLC; icotinib.

SCHEME 1

Synthetic route to icotinib-1,2,3-triazole derivatives 3.

FIGURE 1

The binding modes of icotinib and compound 3l with EGFR (PDB: 1M17) (A) The binding mode of icotinib in the ATP binding site of EGFR. The AutoDock score for icotinib is −7.07 (B) The binding mode of 3l in the ATP binding site of EGFR. The AutoDock score for 3l is −9.5.

FIGURE 2

Carboxyfluorescein succinimidyl ester dilution assay and the soft-agar assay results (A) and (B) The results of CSFE. The proliferation inhibition of 3l and icotinib on KYSE450 and KYSE450TR cells compared with cells treated with 0.1% DMSO (C) and (D) The results of soft-agar. The proliferation inhibition of 3l and icotinib on KYSE450 and KYSE450TR cells. Unpaired Student’s t test was used in (C) and (D). *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent the mean ± SD.

FIGURE 3

The result of apoptosis and cell cycle in ESCC cell lines induced by 3l and icotinib (A) and (B) The cell cycle of KYSE450 and KYSE450TR cells by 3l and icotinib, compared with cells treated with 0.1% DMSO (C) and (D) The apoptosis of KYSE450 and KYSE450TR cells by 3l and icotinib, compared with cells treated with 0.1% DMSO. Unpaired Student’s t test was used. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent the mean ± SD.

FIGURE 4

Regulation of cancer cells proliferation due to the binding of 3l with wild-type EGFR. (A) Binding sensorgrams for 3l interaction with immobilized wild-type of EGFR. The K _D(M) value between 3l and wild-type EGFR is 8.23 × 10^–6 (B) Binding sensorgrams for icotinib interaction with immobilized wild-type of EGFR. The K _D(M) value between icotinib and wild-type EGFR is 3.60 × 10^–5 (C) The influence of icotinib and 3l on the EGFR-RAS-Raf-MAPK pathway in KYSE450 and KYSE450TR cells.

FIGURE 5

Compound 3l inhibits tumor growth of cell-line-derived xenografts in ESCC. (A) Tumor images from the indicated groups were shown (B) Tumor sizes were monitored every 3 days for more than two continuous weeks (14th days, Control VS. 3l, n = 8,F = 0.857, t = 5.670, p = 0.004) (C) Tumor weight, liver weight and spleen weight were measured at the end of the experiment (Tumor weight, Control VS. 3l, n = 8, F = 0.033, t = 8.386, p = 0.003) (D) Ki-67 expression in harvested xenograft tissues were assessed by immunofluorescence. Representative photographs in different groups were shown (E) The statistical analysis for the immunofluorescence images was shown (Control VS. 3l, n = 8, F = 0.210, t = 10.224, p = 0.002) (F) P-ERK (Tyr202/204) expression in harvested xenograft tissues were assessed by immunohistochemistry. Representative photographs in different groups were shown (G) The statistical analysis for the immunohistochemistry images was shown (Control VS. 3l, n = 8, F = 0.09, t = 11.285, p = 0.003). Unpaired Student’s t test was used in (B,C,E and G). *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent the mean ± SD.

FIGURE 6

The proposed action mechanism oficotinib-triazole derivativesin ESCC cells. Icotinib-triazole derivatives inhibits cell proliferation, induces apoptosis and cell cycle arrest of ESCC cells via EGFR-mediated mitogen-activated protein kinase (MAPK) pathway, leading to the inactivation of RAS-Raf-MAPK pathway.