ZAK is required for doxorubicin, a novel ribotoxic stressor, to induce SAPK activation and apoptosis in HaCaT cells

Abstract

Doxorubicin is an anthracycline drug that is one of the most effective and widely used anticancer agents for the treatment of both hematologic and solid tumors. The stress-activated protein kinases (SAPKs) are frequently activated by a number of cancer chemotherapeutics. When phosphorylated, the SAPKs initiate a cascade that leads to the production of proinflammatory cytokines. Some inhibitors of protein synthesis, known as ribotoxic stressors, coordinately activate SAPKs and lead to apoptotic cell death. We demonstrate that doxorubicin effectively inhibits protein synthesis, activates SAPKs, and causes apoptosis. Ribotoxic stressors share a common mechanism in that they require ZAK, an upstream MAP3K, to activate the pro-apoptotic and proinflammatory signaling pathways that lie downstream of SAPKs. By employing siRNA mediated knockdown of ZAK or administration of sorafenib and nilotinib, kinase inhibitors that have a high affinity for ZAK, we provide evidence that ZAK is required for doxorubicin-induced proinflammatory and apoptotic responses in HaCaT cells, a pseudo-normal keratinocyte cell line, but not in HeLa cells, a cancerous cell line. ZAK has two different isoforms, ZAK-α (91 kDa) and ZAK-β (51 kDa). HaCaT or HeLa cells treated with doxorubicin and immunoblotted for ZAK displayed a progressive decrease in the ZAK-α band and the appearance of ZAK-β bands of larger size. Abrogation of these changes after exposure of cells to sorafenib and nilotinib suggests that these alterations occur following stimulation of ZAK. We suggest that ZAK inhibitors such as sorafenib or nilotinib may be effective when combined with doxorubicin to treat cancer patients.

Figure 1

Effect of ZAK knockdown on doxorubicin-induced apoptosis, activation of SAPKs, and inhibition of protein synthesis. (A) HaCaT cells were exposed to ZAK-specific siRNA (zak) or scrambled siRNA (scr) prior to treatment for 24 h with vehicle or doxorubicin (5, 25 or 50 µM), as described in Materials and Methods. Lysates were collected and subjected to SDS-PAGE followed by western blot analysis with the antibodies indicated. (B) HaCaT cells grown on glass coverslips were exposed to scrambled siRNA (i and iii) or ZAK-specific siRNA (ii and iv) prior to treatment for 24 h with vehicle (i and ii) or 25 µM doxorubicin (iii and iv). Cells were exposed to bisbenzimide to examine fluorescent nuclear staining as described in Materials and Methods. (C) HaCaT cells were exposed to scrambled siRNA (scr) or ZAK-specific siRNA (zak) sequence #1 from (A) or sequence #2 prior to treatment for 24 h with vehicle or 25 µM doxorubicin, as described in Materials and Methods. Lysates were collected and subjected to SDS-PAGE followed by western blot analysis with the antibodies indicated. (D) HaCaT cells were treated in triplicate for 6, 12 or 24 hours with either vehicle or 1, 2.5, 10 or 25 µM of doxorubicin. Cells were pulse-labeled with [³H]-leucine for the final 30 minutes of treatment.

Figure 2

Inhibition of doxorubicin-induced activation of SAPKs by pre-treatment with emetine. (A) HaCaT cells were pretreated with vehicle (ii) or emetine (100 µg/mL; i and iii) for 30 minutes prior to stimulation with doxorubicin (0.5 mM; ii and iii) for 15, 30, 60 or 120 min. Lysates were collected and submitted to SDS-PAGE, followed by western blot analysis with the antibodies as indicated. (B) HaCaT cells were treated in triplicate for 1, 2, 3 or 4 hours with vehicle or 0.5 mM doxorubicin. Cells were pulse-labeled with [³H]-leucine for the final 30 minutes of treatment. (C) HaCaT cells were pretreated with vehicle (ii) or emetine (100 µg/mL; i and iii) for 30 minutes prior to stimulation as indicated with CdCl₂ (100 µM; ii and iii) for 15, 30, 60 or 120 min. Lysates were collected and subjected to SDS-PAGE, followed by western blot analysis with the antibodies as indicated.

Figure 3

Effect of inhibitors on doxorubicin- and daunorubicin-induced apoptosis and activation of SAPKs. HaCaT cells were pretreated for 30 minutes with vehicle or inhibitors. Twelve hours later an additional dose of inhibitor was added to each well. Twenty-four hours after the addition of doxorubicin or daunorubicin, lysates were collected and subjected to SDS-PAGE followed by western blot analysis with the antibodies as indicated. (A) HaCaT cells were pretreated with vehicle (C), sorafenib (S; 1 µM), or nilotinib (N; 1 µM) for 30 minutes, followed by treatment with doxorubicin (10 or 25 µM) for 24 h. (B) HaCaT cells were pretreated with vehicle (C), sorafenib (S; 1 µM), or nilotinib (N; 1 µM) for 30 minutes, followed by treatment with daunorubicin (2.5, 10 or 25 µM) for 24 h. (C) HaCaT cells were pretreated with vehicle (C), SB 203580 (SB; 10 µM), SP 600125 (SP; 20 µM), both (SB SP), or zVAD-fmk (Z; 25 µM) for 30 minutes, followed by treatment with doxorubicin (25 µM) for 24 h.

Figure 4

Effect of ZAK knockdown, sorafenib or nilotinib on doxorubicin-induced apoptosis in HeLa cells. Following exposure to 10 or 25 µM doxorubicin, HeLa cell lysates were collected and subjected to SDS-PAGE, followed by western blot analysis with the antibodies as indicated. (A) Cells were pretreated with vehicle (C), sorafenib (S; 1 µM), or nilotinib (N; 1 µM) for 30 minutes, followed by treatment with doxorubicin for 24 h. At 12 h an additional dose of vehicle or inhibitors was added to each well. (B) Cells were exposed to scrambled siRNA (scr) or ZAK-specific siRNA (zak) as described in Materials and Methods, followed by treatment for 24 h with vehicle or doxorubicin.

Figure 5

Effect of inhibition of ZAK, p38 MAPK or JNK on doxorubicin-induced degradation or modification of ZAK. (A) HaCaT cells were exposed to 25 µM doxorubicin and were harvested at 4 h intervals. Lysates were subjected to SDS-PAGE, followed by western blot analysis with the antibodies as indicated. (B) HaCaT cells were exposed to 25 µM doxorubicin. Eight hours later, 5 µM MG-132 or vehicle was added to the corresponding wells. Treatment continued for an additional 12 hours, at which time cells were harvested. Lysates were subjected to SDS-PAGE, followed by western blot analysis with the antibodies as indicated. (C) Western blot analysis with ZAK antibody on cell lysates from Figure 3A. (D) Western blot analysis with ZAK antibody on cell lysates from Figure 3C.