*2.4. DAPK1*/*TAp63-Mediated Autophagy Regulates Apoptotic Death in Drug-Resistant Ovarian Cancer Cells after Consecutive Treatment with Gliotoxin and Paclitaxel*

DAPK1 and p53 mutually induce apoptosis in cancer cells (6, 11). To determine the association between DAPK1 and TAp63 in inducing apoptosis in CaOV3/PTX\_R and SKOV3/PTX\_R cells after sequential treatment with gliotoxin and paclitaxel, we investigated the effect of DAPK1 on TAp63 expression, autophagosome-related molecule levels, and mitochondrial membrane potential changes. DAPK1-knockdown CaOV3/PTX\_R and SKOV3/PTX\_R cells exhibited suppressed TAp63, XAF-1, LC3-I/II, and Beclin-1 expression and downregulated MDR1 and MRP1-3 expression after sequential treatment with gliotoxin and then paclitaxel (Figure 4A). In addition, DAPK1 gene silencing using siRNA prevented the cleavage of caspase-9, caspase-3, and PARP (Figure 4B) as well as the depolarization of mitochondria membranes induced by treating paclitaxel-resistant ovarian cancer cells with gliotoxin followed by paclitaxel (Figure 4C). We finally investigated the connection between the DAPK1-TAp63 signaling pathway and autophagy-related cell death using 3-methyladenine (3-MA), an autophagy inhibitor. Pre-exposure of CaOV3/PTX\_R and SKOV3/PTX\_R cells to 3-MA had no effect on the expression of DAPK1, TAp63, and XAF1 after subsequent treatment with gliotoxin and paclitaxel (Figure 5A), whereas multidrug resistant-associated protein levels remained low (Figure 5A). Furthermore, 3-MA efficiently blocked the activation of the caspase-dependent apoptotic pathway and the depolarization of mitochondria membranes after sequential treatment with gliotoxin followed by paclitaxel (Figure 5B,C). These results suggest that DAPK1/TAp63-mediated autophagy is one of the key downstream target pathways that induce apoptosis in drug-resistant ovarian cancer cells and demonstrate that multidrug resistant-associated protein levels are regulated in an autophagy-independent manner after sequential treatment with gliotoxin followed by paclitaxel.

**Figure 3.** Pre-exposure to gliotoxin followed by paclitaxel induces caspase-dependent apoptosis in drug-resistant ovarian cancer cells by upregulating TAp63 expression. (**A**,**B**) Cells were seeded into 6-well plates (1.5 <sup>×</sup> 10*<sup>5</sup>* cells/well), pre-treated with GTX (5 <sup>μ</sup>M) for 8 h and then treated with PTX (100 nM) for an additional 48 h. For comparison, untreated control cells were cultured with media in the presence of DMSO. Total protein was subjected to Western blot analysis with the indicated antibodies. The cells treated with GTX followed by PTX upregulated the expression of DAPK1 and TAp63. β-actin served as an internal control. (**C**,**D**) Cells were transfected with either empty vector pcDNA3.1 or TAp63 expression vector. (**C**) Transfection efficiency was determined by immunoblot using TAp63 and FLAG antibodies. Whole cell lysates were analyzed by Western blotting using the indicated antibodies. Overexpression of TAp63 downregulated the levels of MDR1 and MRP1-3. (**D**) Percentages of apoptotic cells were analyzed by annexin-V/7-AAD staining. The number of late-stage apoptotic cells (annexin-V+/7-AAD+) was calculated by flow cytometry. (**E**,**F**) Cells (1.5 <sup>×</sup> <sup>10</sup>*5*/well) were pre-treated with GTX (5 μM) for 8 h and then treated with PTX (100 nM) for an additional 24 h. Then, the cells were transfected with 200 nM siRNA against TAp63 or control. Cells were used for further experiments 40 h after transfection. The cells were analyzed by Western blotting with the indicated antibodies. Targeted inhibition of TAp63 suppressed the expression of autophagosome-related LC3-I/II and Beclin-1 (E) and prevented the upregulation of activated caspase-9 (active p37/35) and caspase-3 (active p19/17) for apoptotic death (**F**). β-actin served as an internal control. The results are representative of three independent experiments.

**Figure 4.** Increased DAPK1 induced by pre-exposure to gliotoxin and paclitaxel upregulates TAp63 expression and autophagy signaling in drug-resistant ovarian cancer cells. The cells (1.5 <sup>×</sup> 10*5*/well) were pre-treated with GTX (5 μM) for 8 h and then treated with PTX (100 nM) for an additional 24 h. Next, cells were transfected with 200 nM siRNA against TAp63 or control. Cells were used for further experiments 40 h after transfection. (**A**,**B**) The cells were analyzed by Western blotting with the indicated antibodies. DAPK1 silencing prevented the activation of downstream target molecules, including transcriptionally active p63 (TAp63), XIAP-associated factor 1 (XAF-1), LC3-I/II, and Beclin-1 as well as the cleavage of caspase-9 (active p37/35) and caspase-3 (active p19/17) by treatment with gliotoxin followed by paclitaxel. β-actin served as an internal control. (**C**) To measure Δψ*<sup>m</sup>* disruption, cells were stained with DiOC6 and analyzed by flow cytometry. Diminished DiOC*<sup>6</sup>* fluorescence (%) indicates Δψ*<sup>m</sup>* disruption. The results are representative of three independent experiments.

**Figure 5.** DAPK1/TAp63-mediated autophagy induction mediates apoptotic death in drug-resistant ovarian cancer cells after continuous treatment with gliotoxin and paclitaxel. (**A**–**C**) To inhibit autophagic signaling, cells (1.5 <sup>×</sup> 10*5*/well) were pre-exposed to 3-methyladenine (3-MA) (10 mM) for 2 h. Cells were pre-treated with GTX (5 μM) for 8 h and then treated with PTX (100 nM) for an additional 48 h. For comparison, untreated control cells were cultured with media in the presence of DMSO. (**A**,**B**) Whole cell lysates were subjected to Western blot analysis using the indicated antibodies. Pretreatment with 3-MA effective prevented the expression of autophagosome-related proteins (LC3-I/II and Beclin-1) and cleaved form of caspase-9 (active p37/35) and caspase-3 (active p19/17), but had no effect on downregulation of MDR-1 and MRP1-3 after treatment with gliotoxin followed by paclitaxel. β-actin served as an internal control. (**C**) To measure Δψ*<sup>m</sup>* disruption, cells were stained with DiOC*<sup>6</sup>* and analyzed by flow cytometry. Diminished DiOC*<sup>6</sup>* fluorescence (%) indicates Δψ*<sup>m</sup>* disruption. The results are representative of three independent experiments.

#### **3. Discussion**

The tumor suppressor p53 promotes autophagy by inducing various autophagy-related genes, including DAPK1, a kinase acting in the early steps of autophagy [14,34]. DAPK1 overexpression also promotes the activation of cell death-associated signaling pathways, including autophagy-related apoptosis [1]. However, DAPK1 expression is frequently downregulated in B cell lymphoma and non-small cell lung cancer through multiple mechanisms, including promoter methylation [35,36]. TAp63, a p53 family member sharing a transactivation domain, has been reported to regulate the same target genes [19,20]. TAp63 not only inhibits cell growth but also prevents cell cycle progression in p53-deficient cancer cells [37]. These reports demonstrate that DAPK1 plays an important role in apoptosis induced by cytotoxic drug treatment and that the TAp63 and/or DAPK-related signaling pathways are promising candidates for controlling cancer growth in certain tumor environments. In this study, treatment with gliotoxin reversed the paclitaxel resistance of drug-resistant ovarian cancer cells through the downregulation of multidrug resistant-associated proteins. In addition, sequential treatment with gliotoxin followed by paclitaxel activated the DAPK1-mediated TAp63 signaling pathway to induce autophagic cell death in paclitaxel-resistant ovarian cancer cells (Figure 6). These

results suggest that monitoring TAp63 and DAPK1 expression level is critical for detecting paclitaxel resistance and deciding whether to use paclitaxel in advanced or recurrent ovarian cancer patients.

**Figure 6.** Schematic diagram of the intracellular signaling mechanism after sequential treatment with gliotoxin followed by paclitaxel in human ovarian cancer cells.

DAPK1 activation by cell death-inducing stimuli promotes apoptosis through the activation of p53-dependent p14/p19ARF tumor suppressor genes [5]. DAPK1 overexpression activated autophagic apoptotic death in a caspase-independent manner in breast and cervical cancer cells expressing wild-type p53 [9]. In contrast, stimulation with TGF-beta resulted in DAPK1-induced mitochondrial damage, leading to caspase-dependent apoptosis in a p53-depleted hepatoma cell line [38]. These contradictory results demonstrate that the role of DAPK1 might require further study to understand the connection with p53 or p53 family proteins in the apoptosis pathway. Although treating CaOV3 and SKOV3 cells with paclitaxel increased p53 expression, sequential exposure to gliotoxin followed by paclitaxel upregulated the levels of DAPK1 and TAp63 but not p53 and TAp73 expression in CaOV3/PTX\_R and SKOV3/PTX\_R cells. Furthermore, gene silencing of DAPK1 using siRNA in CaOV3/PTX\_R and SKOV3/PTX\_R cells prevented autophagy induction, caspase activation, and mitochondrial membrane disruption, as well as TAp63 activation. These results suggest that DAPK1 contributes to TAp63 activation to induce autophagic cell death in paclitaxel-resistant ovarian cancer cells after consecutive treatment with gliotoxin and paclitaxel. However, the precise association based on the molecular mechanism of DAPK1 and TAp63 in various cancer environments still needs to be investigated.

Cells that survive previous chemotherapy obtain resistance to several anticancer drugs through their development of various defense mechanisms, including promoting drug efflux capability, altering drug metabolism, and changing drug targets [39]. Inactivation of p53 or mutant p53 in cancer cells decreases drug accumulation through the upregulation of multidrug-resistance protein (MRP1), which mediates ATP-dependent drug efflux [40]. Although exposure to gliotoxin induces mitochondrial membrane disruption and p53-dependent apoptotic cell death in adriamycin-resistant non-small cell lung cancer cells [33], the acquired mechanism to overcome the cytotoxic effects of chemotherapeutic drugs might be very diverse in cell type- or tumor environmental-dependent manners. In addition, the contribution of other p53 family proteins in the absence of wild-type p53 to overcome anticancer drugs is still unclear. Sequential treatment with gliotoxin, followed by paclitaxel increased the level of TAp63 in paclitaxel-resistant ovarian cancer cells. Furthermore, forced expression of TAp63 by transfection with a TAp63-containing plasmid reduced the expression of multidrug resistant-associated proteins (MDR1 and MRP1-3). These results suggest that TAp63 also plays an important role in modulating drug resistance without wild-type p53.

Although pre-exposure to 3-MA before sequential treatment efficiently blocked cleaved caspase-9, caspase-3, and PARP generation and prevented mitochondrial membrane disruption, pretreatment with 3-MA still inhibited multidrug resistant-associated protein levels but failed to attenuate DAPK1-TAp63 signaling pathway activation. These results suggest that the DAPK1-TAp63 pathway controls autophagy induction and drug-resistant protein expression in an independent manner to promote the apoptotic pathway after sequential treatment with gliotoxin, followed by paclitaxel (Figure 6).

Taken together, our results suggest that gliotoxin might be a promising agent to control advanced or recurrent ovarian cancer in clinical situations by reducing paclitaxel resistance. Our data also demonstrate that DAPK1 and TAp63 levels could be used as diagnostic or determining factors of drug resistance before starting repeated chemotherapy against ovarian cancer.
