*2.3. Pre-Exposure to Gliotoxin Followed by Paclitaxel Upregulates the TAp63 Expression, Leading to the Caspase-Dependent Apoptosis in Drug-Resistant Ovarian Cancer Cells*

We next investigated the underlying signaling pathway to determine the role and relationship of DAPK1 and p53 family proteins. Although p53 expression was upregulated in CaOV3 and SKOV3 cells after paclitaxel stimulation (Figure 3A), exposure to gliotoxin or paclitaxel failed to induce p53 expression in CaOV3/PTX\_R and SKOV3/PTX\_R cells (Figure 3B). In contrast, cells treated with gliotoxin followed by paclitaxel had increased expression of DAPK1 and TAp63, a p53 family member (Figure 3B). To determine the role of TAp63 in the apoptotic death of CaOV3/PTX\_R and SKOV3/PTX\_R cells, we transfected TAp63-expressing plasmids into drug-resistant ovarian cancer cells. The expression of drug resistance-associated proteins was prominently lower in CaOV3/PTX\_R and SKOV3/PTX\_R cells with forced TAp63 expression than in cells transfected with empty vector (Figure 3C). Furthermore, TAp63 overexpression led to apoptosis in paclitaxel-resistant ovarian cancer cells after treatment with paclitaxel (Figure 3D). Sequential treatment with gliotoxin followed by paclitaxel induced the expression of autophagosome-related molecules (LC3-I/II and Beclin-1) and the apoptosis-related protein, Bax (Figure S2). Although TAp63 gene silencing had no effect on DAPK1 expression, reduced TAp63 expression prevented the induction of XIAP-associated factor 1 (XAF1), LC3-I/II, and Beclin-1, as well as the reductions in MDR1 and MRP1-3 expression, after sequential treatment with gliotoxin and paclitaxel (Figure 3E). Targeted inhibition of TAp63 also prevented the expression of cleaved and activated caspase-9, caspase-3, and PARP in CaOV3/PTX\_R and SKOV3/PTX\_R cells after treatment with gliotoxin followed by paclitaxel (Figure 3F). These results suggest that TAp63 expression plays an important role in enhancing apoptosis through the downregulation of multidrug resistant-associated proteins.
