Enzastaurin inhibits tumours sensitive and resistant to anti-EGFR drugs

Abstract

We investigated the antitumour effect and ability to overcome the resistance to anti-EGFR drugs of enzastaurin, an inhibitor of VEGFR-dependent PKCbeta signalling. Enzastaurin was evaluated alone and in combination with the EGFR inhibitor gefitinib, on growth and signalling protein expression in human cancer cells sensitive and resistant to anti-EGFR drugs, both in vitro and in nude mice. We demonstrated the marked inhibitory activity of enzastaurin against GEO colon and PC3 prostate cancer cells and their gefitinib-resistant counterparts GEO-GR and PC3-GR, accompanied by inhibition of pAkt and its effector pp70S6K, pGSK3beta and VEGF expression and secretion. Moreover, enzastaurin showed a cooperative effect with gefitinib in parental and in gefitinib-resistant cells. Remarkably, these results were confirmed in vivo, where enzastaurin showed antitumour activity and cooperativity with gefitinib in mice grafted with GEO and GEO-GR tumours, incrementing their median survival and inhibiting the aforesaid protein expression and secretion in tumour specimens. In conclusion, enzastaurin by interfering with signalling proteins implicated in EGFR drug resistance markedly cooperates with gefitinib in sensitive and gefitinib-resistant tumours, thus overcoming and reverting such resistance and providing a rational basis for its development in patients resistant to anti-EGFR drugs.

Figure 1

GEO, GEO-CR, GEO-GR, PC3, PC3-GR and MDA-468 cells were treated with enzastaurin at doses of drug ranged from 0.1 to 5 μM. Data are expressed as the number of colony formation. Data represent the average of triplicate determinations of at least two experiments.

Figure 2

(A) Lane 1, GEO cells untreated; lane 2, GEO cells treated with enzastaurin; lane 3, GEO-GR cells untreated; lane 4, GEO-GR cells treated with enzastaurin; lane 5, PC3 cells untreated; lane 6, PC3 cells treated with enzastaurin; lane 7, PC3-GR cells untreated; lane 8, PC3-GR cells treated with enzastaurin. Cell lysates treated in vitro on days 0 and 2 were collected on day 5. Bars, s.d. (B) ELISA assay for VEGF was done on total lysates from human cancer cell lines treated with 1 μM enzastaurin for 2 days. Lane 1, GEO; lane 2, GEO-CR; lane 3, GEO-GR; lane 4, PC3; lane 5, PC3-GR; lane 6, MDA-468. (C) ELISA assay for VEGF was done on conditioned medium collected from the same cell lines. Data are the average of two different experiments, each performed in triplicate; bars, s.d. Results were reported as pg per mg protein.

Figure 3

(A and B) Effect of enzastaurin and gefitinib, alone and in combination, on the soft agar growth of GEO-GR and PC3-GR cells. Growth inhibition results are expressed as the percentage of the number of colonies developed in each of the different treatment wells compared with the absolute number of colonies developed in the untreated control group. Data represent the average of at least two different experiments run in triplicate. (C and D) Synergistic effect of enzastaurin and gefitinib in combination on GEO-GR and PC3-GR cell growth inhibition. The data represent the plot of CIs, a quantitative measure of the degree of combination treatment for a given end point of the inhibition effect. The CI values of <1, 1 and >1 indicate synergy, additivity and antagonism, respectively. Each point is the mean of at least three different replicate experiments.

Figure 4

Cooperative effect of enzastaurin and gefitinib on tumour growth and survival of mice bearing human colon cancer xenografts GEO (A and B) and GEO-GR (C and D). (A–C) After 7 days following tumour injection, 10 mice were randomised to receive the treatment. The Student's t-test was used to compare tumour sizes among different treatment groups at day 56 following cell injection. Statistically significant differences were observed for enzastaurin vs control (two-sided P<0.0001), enzastaurin+gefitinib vs control (two-sided P<0.0001) and vs enzastaurin (two-sided P<0.0001). Bars, s.d. (B–D) Median survival in GEO (B) and GEO-GR (D) xenografts.

Figure 5

(A) Western blotting was performed on total lysates from tumour specimens of two mice of GEO-GR killed on day 25 and treated as in (C). Lane 1, untreated control; lane 2, gefitinib; lane 3, enzastaurin; lane 4, gefitinib plus enzastaurin. (B and C) ELISA assays for hVEGF were performed on total lysates from tumour specimens (B) and on serum (C) of two mice of GEO-GR killed on day 25.