The important roles of RET, VEGFR2 and the RAF/MEK/ERK pathway in cancer treatment with sorafenib

Abstract

Aim: To elucidate the roles of receptor tyrosine kinases RET and VEGFR2 and the RAF/MEK/ERK signaling cascade in cancer treatment with sorafenib.

Methods: The cell lines A549, HeLa, and HepG2 were tested. The enzyme activity was examined under cell-free conditions using 384-well microplate assays. Cell proliferation was evaluated using the Invitrogen Alarmar Blue assay. Gene expression was analyzed using the Invitrogen SYBR Green expression assays with a sequence detection system. Protein expression analysis was performed using Western blotting.

Results: Sorafenib potently suppressed the activities of cRAF, VEGFR2, and RET with IC(50) values of 20.9, 4 and 0.4 nmol/L, respectively. Sorafenib inhibited cRAF, VEGFR2, and RET via non-ATP-competitive, ATP-competitive and mixed-type modes, respectively. In contrast, sorafenib exerted only moderate cytotoxic effects on the proliferation of the 3 cell lines. The IC(50) values for inhibition of A549, HeLa, and HepG2 cells were 8572, 4163, and 8338 nmol/L, respectively. In the 3 cell lines, sorafenib suppressed the cell proliferation mainly by blocking the MEK/ERK downstream pathway at the posttranscriptional level, which in turn regulated related gene expression via a feed-back mechanism.

Conclusion: This study provides novel evidence that protein kinases RET and VEGFR2 play crucial roles in cancer treatment with sorafenib.

Figure 1

Sorafenib inhibits kinase activity of cRAF, RET, and VEGFR2 with different IC₅₀ under cell-free condition.

Figure 2

Double-reciprocal plot of kinetic data from assays of KDR, RET, and cRAF protein kinase activity at different concentrations of sorafenib. ATP concentrations in the reaction mixture varied from 1/3 ATP K_m to 81 fold ATP K_m. Sorafenib concentrations are from 100 μmol/L to 1.6 nmol/L with 3-fold dilution and the concentration of peptides were kept constant at 2 μmol/L. V is picomoles of phosphate/60 min. Double-reciprocal plot shows the linear regression alignments are converge on Y axis, which means sorafenib is an ATP competitive compound on KDR, RET, which is consistent with reported. The IC₅₀ of RET, KDR are 0.4 nmol/L, 4 nmol/L in sequence.

Figure 3

Sorafenib moderately inhibited proliferation of A549, HeLa and HepG2 cells. Sorafenib was added to A549, HeLa, and HepG2 cells, and cultured for 72 h in culture medium, meanwhile, DMSO was added to controls. Sorafenib displayed moderate cytotoxicity to cell proliferation dose-dependently.

Figure 4

Sorafenib affected RET and VEGFR2 gene expression in A549, HeLa, and HepG2 cells. Three cell lines were treated by sorafenib with two concentration gradients, 1 and 5 μmol/L, and then collected after 2, 4, and 8 h. Cells without sorafenib treatment were as the controls (0 h). Total mRNA was extracted and quantified to be used in RT-PCR assays. Ratio for mRNA content in treatment group compared with controls was calculated. (A) Sorafenib up-regulated RET gene expression and down-regulated VEGFR2 gene expression in A549 cells. (B) Sorafenib up-regulated RET gene expression and down-regulated VEGFR2 gene expression in HeLa cells which was similar to that in A549 cells. (C) Sorafenib down-regulated RET gene expression and up-regulated VEGFR2 gene expression in HepG2 cells which opposite to that in the above two cell lines.

Figure 5

Sorafenib affected the phosphorylation of receptor tyrosine kinase RET and VEGFR2, as well as MEK/ERK kinases signaling cascades in three cell lines. Three cell lines were treated by sorafenib with two concentration gradients, 1 and 5 μmol/L/L, and then collected after 2, 4, and 8 h, cells without sorafenib treatment were as the controls (0 h). Total proteins were extracted and quantified to be used in Western blot assays. (A) Sorafenib inhibited RET and VEGFR2 phosphorylation dose-dependently while activated MEK and ERK phosphorylation in A549 cells. (B) Sorafenib also inhibited RET and VEGFR2 phosphorylation, and slightly activated MEK and ERK phosphorylation in HeLa cells. (C) Sorafenib activated the phosphorylation of RET, VEGFR2, and MEK, but inhibited ERK phosphorylation in HepG2 cells.