**1. Introduction**

Death-associated protein kinase-1 (DAPK1) is a Ca<sup>2</sup>+/calmodulin (CaM)-regulated serine/threonine kinase that mediates cell death [1]. Downregulation of DAPK1 by methylation in its promoter region is detected in various cancers, including pancreas, lung, and head and neck. Low DAPK1 expression in these tumors is closely related to frequent lymph node metastasis and poor clinical outcomes [2–4]. Overexpression or activation of DAPK1, as a tumor suppressor, is involved in cell death. Ectopic DAPK expression induces p53-dependent apoptosis in mouse embryo fibroblasts [5], and tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) induce DAPK1 expression through inhibiting NF-κB activity [6]. DAPK1 activation not only requires cell cycle arrest and caspase-dependent apoptosis [7] but also contributes to autophagic cell death by reducing the interaction between Beclin-1 and Bcl-2 and Bcl-XL [8]. Furthermore, DAPK-1 induces caspase-independent cell death through activating autophagosome formation [9]. However, DAPK1 sometimes antagonizes apoptosis in a cell-type-dependent manner. DAPK1 depletion using antisense DAPK1 cDNA promotes caspase-mediated apoptosis via TNF [10], and DAPK1 plays an essential role in the proliferation of

p53-mutant estrogen receptor-negative breast cancer cells [11]. These results demonstrate that the role and molecular mechanism of DAPK1 in human cancers are not clearly understood.

Tumor suppressor p53 expression responds to various types of cellular damage and stress, including oncogenic stimuli [12,13]. Cells expressing wild-type p53 experience DAPK1 upregulation after stimulation with anticancer drugs or UV exposure [14]. In contrast, DAPK1 overexpression promotes p53 expression, resulting in the suppression of oncogenic transformation [5]. p53 mutations that reduce or abolish its function are closely associated with anticancer drug resistance in various cancers [15,16], whereas mutant p53 occurrence in ovarian cancer has no effect on apoptotic death induced by paclitaxel [17]. Ovarian cancer co-expressing p53 and Bcl-2 has been shown to have the best response to paclitaxel chemotherapy [18]. These contradictory results suggest that p53 and DAPK1 influence several different molecular pathways to induce cancer cell death, and the mutual relationship between DAPK1 and p53 is dependent on cell type and cell conditions.

The p53 family is composed of three homologous proteins, p53, p63, and p73. These proteins share essential structural domains and have similar cellular functions in proliferation, differentiation, tumorigenesis, and death [19,20]. Transcriptionally active p63 (TAp63), a p53 isoform, plays a critical role in not only intracellular fatty acid generation [21] but also tumor suppression [22] and metastasis prevention [23]. We have previously reported that TAp63 activation induces apoptosis in Epstein-Barr virus (EBV)-transformed B cells after treatment with baicalein [24], and TAp63 expression in TLR4-stimulated colon cancer cells promotes fatty acid-mediated metastasis [25]. However, the precise relationship between DAPK1 and TAp63 in modulating apoptosis in cancer is unclear, and the autophagy-related signaling pathway regulated by TAp63 in cancer needs to be investigated.

Gliotoxin, a secondary metabolite of marine fungus *Aspergillus fumigatus*, is not only a member of the epipolythiodioxopiperazine family but also characterized by a disulfide bond across a piperazine ring and an aromatic amino acid [26,27]. Treatment with gliotoxin of mouse immune cells inhibits nuclear factor kappa B (NF-κB), resulting in the downregulation of inflammatory genes activation [28]. Reactive oxygen species (ROS) induced by gliotoxin contribute to suppression of NF-κB, leading to the apoptosis of human fibrosarcoma cell line (HT1080) [29]. Gliotoxin induces apoptosis in various human cancers cells, including uterine cervix cancer cell line (Hela), chondrosarcoma cell line (SW1353), chronic lymphocytic leukemia cell, and breast cancer cell line (MCF-7) [30–32]. Recently, it has been reported that gliotoxin treatment induces apoptotic death in doxorubicin-resistant lung cancer cells through disrupting mitochondrial function and activating p53 downstream target molecules [33]. However, the expression and specific role of DAPK1 in ovarian and paclitaxel-resistant ovarian cancer cells after treatment with gliotoxin have not been studied. In this study, we investigated whether DAPK1 regulates apoptotic death in paclitaxel-resistant ovarian cancer cells and examined the relationship between DAPK1 and p53 family proteins in inducing autophagic cell death after treatment with gliotoxin.
