Small molecule kinase inhibitors block the ZAK-dependent inflammatory effects of doxorubicin

Abstract

The adverse side effects of doxorubicin, including cardiotoxicity and cancer treatment-related fatigue, have been associated with inflammatory cytokines, many of which are regulated by mitogen-activated protein kinases (MAPKs). ZAK is an upstream kinase of the MAPK cascade. Using mouse primary macrophages cultured from ZAK-deficient mice, we demonstrated that ZAK is required for the activation of JNK and p38 MAPK by doxorubicin. Nilotinib, ponatinib and sorafenib strongly suppressed doxorubicin-mediated phosphorylation of JNK and p38 MAPK. In addition, these small molecule kinase inhibitors blocked the expression of IL-1β, IL-6 and CXCL1 RNA and the production of these proteins. Co-administration of nilotinib and doxorubicin to mice decreased the expression of IL-1β RNA in the liver and suppressed the level of IL-6 protein in the serum compared with mice that were injected with doxorubicin alone. Therefore, by reducing the production of inflammatory mediators, the inhibitors identified in the current study may be useful in minimizing the side effects of doxorubicin and potentially other chemotherapeutic drugs.

Figure 1. MAPK activation and gene expression in doxorubicin-treated BMDM. BMDM were plated onto 12-well plates. After serum deprivation for 0.5 h, BMDM were untreated, treated with 5 µM of doxorubicin for 2 h, then washed away and replaced by medium, or treated continuously with 5 µM of doxorubicin for 12 or 24 h. (A) Western blot of cell lysates using antibodies against phospho-JNK, phospho-p38 MAPK or total p38 MAPK. (B) Measurement of gene expression in cells after identical treatments using real-time RT-PCR. Mean values ± SD are shown. ** p < 0.01; *** p < 0.001 compared with values corresponding to wild-type.

Figure 2. MAPK activation in WT, ZAK^+/− and ZAK^−/− BMDM. Serum-deprived BMDM were untreated or treated with 500 μM doxorubicin for 3 h. (A) Western blot of cell lysates from ZAK^−/− (lanes 1 and 4), ZAK^+/− (lanes 2 and 5) and WT (lanes 3 and 6) mice. (B) Measurement of gene expression in cells from four individual ZAK^−/− mice (lanes 1–4), one ZAK^+/− mouse (lane 5) and one WT mouse (lane 6) using real-time RT-PCR. Mean values ± SD are shown. *** p < 0.001 compared with values of the corresponding control treatments.

Figure 3. Effect of small molecule kinase inhibitors on doxorubicin-mediated MAPK phosphorylation, gene expression and cytokine secretion. (A) Western blot of cell lysates from BMDM treated with 500 μM doxorubicin for 3 h in the presence or absence of 1 µM nilotinib, ponatinib or sorafenib. B, measurement of gene expression in cells untreated or treated continuously with 5 µM doxorubicin for 12 h in the presence or absence of 1 µM nilotinib, ponatinib or sorafenib using real-time RT-PCR. (C) Measurement of gene expression in cells untreated or treated with 5 µM of doxorubicin for 2 h in the presence or absence of 1 µM nilotinib, ponatinib or sorafenib then washed away and replaced by medium in the presence or absence of 1 µM nilotinib, ponatinib or sorafenib for 10 h. (D) Cytokine measurement of the culture supernatant from (B) using bead-based multiplex assay. (E) Cytokine measurement of the culture supernatant from (C). Mean values ± SD are shown. * p < 0.005; ** p < 0.01; *** p < 0.001 compared with values corresponding to treatment with doxorubicin alone.

Figure 4. Effect of nilotinib in doxorubicin-mediated RNA expression and cytokine production in mice. Mice were treated with 0.1 mL of vehicle or nilotinib (75 mg/kg) daily for 6 d by oral gavage. On the sixth day, mice were also injected with saline or doxorubicin (25 mg/kg). Sixteen hours post-injection, blood was collected by cardiac puncture and livers were snap-frozen for RNA isolation. (A) Measurement of gene expression in liver samples using real-time PCR. (B) Cytokine measurement of serum samples using bead-based multiplex assay. Mean values ± SEM are shown. * p < 0.005; ** p < 0.01.