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. 2011 Nov 8;105(10):1542-53.
doi: 10.1038/bjc.2011.400. Epub 2011 Oct 4.

Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin

E Giovannetti  1 , P A Zucali, Y G Assaraf, L G Leon, K Smid, C Alecci, F Giancola, A Destro, L Gianoncelli, E Lorenzi, M Roncalli, A Santoro, G J Peters
Affiliations

Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin

E Giovannetti et al. Br J Cancer. .

Abstract

Background: Although pemetrexed, a potent thymidylate synthase (TS) inhibitor, enhances the cytoytoxic effect of platinum compounds against malignant pleural mesothelioma (MPM), novel combinations with effective targeted therapies are warranted. To this end, the current study evaluates new targeted agents and their pharmacological interaction with carboplatin-pemetrexed in human MPM cell lines.

Methods: We treated H2052, H2452, H28 and MSTO-211H cells with carboplatin, pemetrexed and targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, enzastaurin and ZM447439) and evaluated the modulation of pivotal pathways in drug activity and cancer cell proliferation.

Results: Vandetanib emerged as the compound with the most potent cytotoxic activity, which interacted synergistically with carboplatin and pemetrexed. Drug combinations blocked Akt phosphorylation and increased apoptosis. Vandetanib significantly downregulated epidermal growth factor receptor (EGFR)/Erk/Akt phosphorylation as well as E2F-1 mRNA and TS mRNA/protein levels. Moreover, pemetrexed decreased Akt phosphorylation and expression of DNA repair genes. Finally, most MPM samples displayed detectable levels of EGFR and TS, the variability of which could be used for patients' stratification in future trials with vandetanib-pemetrexed-carboplatin combination.

Conclusion: Vandetanib markedly enhances pemetrexed-carboplatin activity against human MPM cells. Induction of apoptosis, modulation of EGFR/Akt/Erk phosphorylation and expression of key determinants for pemetrexed and carboplatin activity contribute to this synergistic interaction, and, together with the expression of these determinants in MPM samples, warrant further clinical investigation.

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Figures

Figure 1
Figure 1
Cytotoxicity and pharmacological interaction of carboplatin, pemetrexed and vandetanib. (A) Representative curves of growth inhibitory effects of carboplatin, pemetrexed, vandetanib and their combinations in H28 cells (for the combinations drug concentrations on the X-axis are referred to pemetrexed). (B) CI fraction affected (FA) plots of the carboplatin–pemetrexed, carboplatin–vandetanib, pemetrexed–vandetanib and carboplatin–pemetrexed–vandetanib combinations in H28 cells. (C) Mean CI values of all the combinations in H28 and H2452 cells. CI values at FA of 0.25, 0.5, 0.75 and 0.9 were averaged for each experiment, and this value was used to calculate the mean between experiments, as described in the Materials and Methods section. Points and columns, mean values obtained from three independent experiments; bars, s.e.m.
Figure 2
Figure 2
Effects of carboplatin, pemetrexed, vandetanib and their combinations on critical determinants of drug activity in H28 cells. (A) Modulation of phosphorylation of EGFR and downstream molecules ERK1/2 and Akt. (B) Modulation of TS, E2F-1, ERCC1, XPD and VEGF mRNA as determined by real-time RT–PCR. (C) Representative blots illustrating the modulation of TS protein expression. (D) Modulation of TS activity. Columns, mean values obtained from three independent experiments; bars, s.e.m. *Significantly different from controls (P<0.05). #Significantly different from pemetrexed-treated cells (P<0.05).
Figure 3
Figure 3
Immunohistochemistry and PCR of MPM samples. (A) Representative examples (original magnification, × 40) from staining of paraffin-embedded MPM samples (36 epithelioid, 7 biphasic and 1 sarcomatoid) for TS (right panels) and EGFR (left panels). TS showed positive cytoplasmic and nuclear staining in most tissue sections (arrows), with intense staining in 8 out of 44 samples, whereas EGFR membrane immunoreactivity (arrows) was detected in 23 out of 44 cases (52.3%): 15.9% specimens showed high expression levels. (B) mRNA expression values of TS and EGFR observed across the cohort of MPM patients, and correlation between TS (C) and EGFR (D) mRNA and protein expression. Values of mRNA and protein expression were calculated as described in the Materials and Methods section. Data were analysed by both Student's t and χ2-test. Columns, mean values; bars, s.d.
Figure 4
Figure 4
Molecular signalling pathways and key determinants involved in the synergistic interaction of vandetanib with pemetrexed and carboplatin. Vandetanib enhanced the growth inhibitory effects of cytotoxic drugs through its pronounced antisignalling effects downstream of EGFR. Furthermore, the modulation of TS and DNA repair genes promotes pemetrexed and carboplatin activity.
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