*2.2. Sequential Treatment with Gliotoxin Followed by Paclitaxel Promotes Apoptotic Death in Paclitaxel-Resistant Ovarian Cancer Cells*

As shown in Figure 1B, treatment with 5 μM GTX not only started to prevent the proliferation of PTX-sensitive SKOV3 cells but also blocked the growth of CaOV3/PTX\_R and SKOV3/PTX\_R cells. Furthermore, exposure to 5 μM GTX reduced in MDR1 and MRP1-3 expression in CaOV3/PTX\_R and SKOV3/PTX\_R cells, but not the induction of active form caspase-9 and caspase-3. We also observed that the exposure to 100 nM paclitaxel for 48 h induced nearly completely blocked the proliferation of PTX-sensitive ovarian cancer cells, whereas the growth rate of CaOV3/PTX\_R and SKOV3/PTX\_R cells was preserved (Figure S1). Based on these results, we next investigated whether

co-treatment with gliotoxin and paclitaxel promotes apoptotic death in drug-resistant ovarian cancer cells. To verify the sensitizing effect of gliotoxin to the anti-cancer drug through reducing MDR1 and MRP1-3 in paclitaxel-resistant ovarian cancer cells, CaOV3/PTX\_R and SKOV3/PTX\_R cells were pre-exposed to gliotoxin (5 μM) for 8 h and then sequentially treated with paclitaxel (100 nM) for 48 h. Consecutive treatment with gliotoxin and paclitaxel significantly prevented CaOV3/PTX\_R and SKOV3/PTX\_R cell growth compared to co-treatment and reverse sequential treatment (Figure 2A). When CaOV3/PTX\_R and SKOV3/PTX\_R cells were treated with gliotoxin, and then paclitaxel, the apoptotic death of chemoresistant ovarian cancer cells was synergistically increased (Figure 2B,C). Furthermore, drug-resistant ovarian cancer cells treated with gliotoxin followed by paclitaxel exhibited activation and cleavage of caspase-9, caspase-3, and PARP (Figure 2D). These results suggest that pre-exposure to gliotoxin reverses paclitaxel resistance in chemoresistant ovarian cancer cells via the induction of apoptotic death by chemotherapeutic agents.

**Figure 2.** Sequential treatment with gliotoxin followed by paclitaxel induces apoptotic death in paclitaxel-resistant ovarian cancer cells. Cells were seeded into 96-well plates (1 <sup>×</sup> 10*<sup>4</sup>* cells/well) or 6-well plates (1.5 <sup>×</sup> 10*<sup>5</sup>* cells/well) and pre-treated with GTX (5 <sup>μ</sup>M) for 8 h followed by PTX (100 nM) for 48 h. For comparison, untreated control cells were cultured with media in the presence of DMSO. (**A**) Cell viability was measured using a Cell Counting Kit-8 assay. The absorbance at 450 nm is presented. n = 3. \**p* < 0.001 (PTX\_R ovarian cancer cells treated with GTX followed by PTX vs. DMSO-treated PTX\_R ovarian cancer cells). (**B**,**C**) To determine the degree of apoptosis, cells were stained with annexin-V-FITC and 7-AAD and analyzed by flow cytometry. Dot-plot graphs show the percentage of viable cells (annexin-V*-* /7-AAD*-* ), early-stage apoptotic cells (annexin-V+/7-AAD*-* ), late-stage apoptotic cells (annexin-V+/7-AAD+), and necrotic cells (annexin-V*-* /7-AAD+). Late-stage apoptotic cells (annexin-V+/7-AAD+) were evaluated by flow cytometry. # *p* < 0.005 (PTX\_R ovarian cancer cells treated with GTX, followed by PTX vs. DMSO-treated PTX\_R ovarian cancer cells). To measure Δψ*<sup>m</sup>* disruption, cells were stained with DiOC*6*. Diminished DiOC*<sup>6</sup>* fluorescence (%) indicates Δψ*<sup>m</sup>* disruption. ## *p* < 0.005 (PTX\_R ovarian cancer cells treated with GTX, followed by PTX vs. DMSO-treated PTX\_R ovarian cancer cells). Each value is expressed as the mean ± SD from three independent experiments (n = 3). (**D**) Whole cell lysates were subjected to Western blot analysis using the indicated antibodies. Sequential treatment with GTX followed by PTX induced the activated caspase-9 (active p37/35) and caspase-3 (active p19/17) in PTX\_R ovarian cancer cells. β-actin served as an internal control. The results are representative of three independent experiments.
