**3. Results**

### *3.1. Characterization of DTX-Resistant PCa Cell Lines*

We used C4-2-DR and DU145-DR cells to analyze the involvement of KIFC1 in DTX resistance. MTT assays were performed to measure cell viability under various concentrations of DTX in the C4-2-DR and DU145-DR cells. The IC50 values of the C4-2-DR and DU145-DR cells were significantly higher than those of the parental DU145 and C4-2 cells, which was consistent with previous results (Figure 1A) [22,23]. We compared the expression of c-PARP, which was used as a marker of apoptosis in the parental and DTX-resistant cell lines. Western blotting showed that the expression of c-PARP and c-caspase-3 was induced by DTX treatment in the parental DU145 and C4-2 cells. On the contrary, the expression of c-PARP was not changed by DTX treatment in DU145-DR and C4-2-DR cells (Figure 1B). These results sugges<sup>t</sup> that the DU145-DR and C4-2-DR cells were resistant to DTX treatment.

### *3.2. KIFC1 is Overexpressed in DTX-Resistant Cell Lines*

To verify whether KIFC1 is involved in DTX resistance, we investigated the expression of KIFC1 in DU145-DR and C4-2-DR cells. Western blotting and qRT-PCR showed that KIFC1 was overexpressed in DU145-DR and C4-2-DR cells compared with the parental DU145 and C4-2 cells at both mRNA and protein levels (Figure 2A,B).

**Figure 1.** Characterization of docetaxel (DTX)-resistant prostate cancer cell lines. (**A**) The dose-dependent effects of DTX on the viability of parental and DTX-resistant cell lines in DU145 and C4-2 cells. The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01. (**B**) Western blotting of c-PARP and c-caspase-3 in parental and DTX-resistant cell lines in DU145 and C4-2 cells in the presence of DTX (10 nM) or vehicle (ethanol). β-actin was used as a loading control. c-PARP: cleaved PARP; c-caspase-3: cleaved caspase-3.

**Figure 2.** KIFC1 is overexpressed in docetaxel (DTX)-resistant cell lines and in a castration-resistant prostate cancer (CRPC) patient. (**A**) Western blotting of KIFC1 in parental and DTX-resistant cell lines. β-actin was used as a loading control. (**B**) qRT-PCR of KIFC1 in parental and DTX-resistant cell lines. The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01.

### *3.3. Inhibition of KIFC1 Induces Apoptosis Pathway and Reverses DTX Resistance In Vitro*

Several studies have shown that KIFC1 is associated with an apoptosis pathway [24,25]. We used RNA interference targeting KIFC1 in DU145-DR and C4-2-DR cells and confirmed the efficiency of KIFC1 knockdown by Western blotting (Figure 3A). Western blotting showed that inhibition of KIFC1 enhanced the expression of Bax2, c-PARP, and c-caspase-3 and reduced the expression of Bcl-2 in DU145-DR and C4-2-DR cells (Figure 3A). Given that KIFC1 was overexpressed in the DTX-resistant cell lines and is involved in the apoptosis pathway, we next analyzed whether the knockdown of KIFC1 improves DTX sensitivity in DU145-DR and C4-2-DR cells. We measured cell viability in DU145-DR and C4-2-DR cells with knockdown of KIFC1 under various concentrations of DTX. We found that downregulation of KIFC1 re-sensitized DU145-DR and C4-2-DR cells to DTX treatment (Figure 3B).

**Figure 3.** Inhibition of KIFC1 induces an apoptosis pathway and reverses docetaxel (DTX) resistance in vitro. (**A**) Western blotting of KIFC1, c-PARP, Bcl-2, Bax, and c-caspase-3 in DU145-DR and C4-2-DR cells transfected with a negative control or two different siRNAs for KIFC1. β-actin was used as a loading control. c-PARP: cleaved PARP; c-caspase-3: cleaved caspase-3 (**B**) The dose-dependent effects of DTX on the viability of DU145-DR and C4-2-DR cells transfected with negative control or two different siRNAs for KIFC1. The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01.

### *3.4. Effect of KIFC1 Inhibitor CW069 on Cell Viability*

A recent study reported that CW069 is a novel and allosteric inhibitor of KIFC1 [26]. To clarify the effect of CW069 on cell viability in PCa, we measured cell viability under various concentrations of CW069 in both parental and DTX-resistant cell lines. CW069 treatment suppressed cell viability in both the parental and DTX-resistant cell lines (Figure 4A). The IC50 values of the DTX-resistant cell lines treated with CW069 were significantly lower than those of the parental cell lines, suggesting that the effect of CW069 on cell viability may depend on the expression of KIFC1. Next, to test whether CW069 could selectively suppress cell viability in cancer cells, we investigated the effect of CW069 in RWPE-1 cells, which is a normal prostate epithelial cell line [27]. Western blotting demonstrated that the expression of KIFC1 was not detected in RWPE-1 cells (Figure 4B). As we expected, CW069 treatment had little effect on cell viability in RWPE-1 cells compared with the DU145 and C4-2 cells (Figure 4C). Furthermore, we performed a cell death ELISA assay to analyze the ability of CW069 to induce apoptotic cell death in RWPE-1, DU145, and C4-2 cells. CW069 treatment had little effect on apoptotic cell death in the RWPE-1 cells but had a significant effect on the DU145 and C4-2 cells (Figure 4D).

**Figure 4.** The effect of the KIFC1 inhibitor CW069 on cell viability (**A**) The dose-dependent effects of CW069 on cell viability in parental and docetaxel-resistant cell lines in DU145 and C4-2 cells. The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01. (**B**) Western blotting of KIFC1 in RWPE-1, DU145, and C4-2 cells. (**C**) The dose-dependent effects of CW069 on cell viability in RWPE-1, DU145, and C4-2 cells. The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01. (**D**) The cell death ELISA in RWPE-1, DU145, and C4-2 cells treated with CW069 (250 μM). The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01.

### *3.5. CW069 Re-Sensitizes DTX-Resistant Cell Lines to DTX Treatment*

As shown in Figure 3B, knockdown of KIFC1 reversed DTX resistance. Therefore, we investigated the effect of combination therapy with DTX and CW069. We measured cell viability under DTX alone or in combination with CW069 in parental and DTX-resistant cell lines. DTX alone had little effect on cell viability in the DTX-resistant cell lines. However, the combination of DTX and CW069 significantly reduced cell viability in the DTX-resistant cell lines (Figure 5A). The cell death ELISA assay showed that the combination of DTX and CW069 led to significant induction of apoptosis compared to DTX alone in the DTX-resistant cell lines (Figure 5B). In addition, we analyzed the dose response for the combination of DTX and CW069 in the DTX-resistant cell lines and calculated the combination index to assess whether the combination of DTX and CW069 is synergistic or additive. A synergistic effect was observed in the DTX-resistant cell lines (Table 1).

**Figure 5.** CW069 re-sensitizes DTX-resistant cell lines to docetaxel (DTX) treatment. (**A**) The effect of the combination of DTX and CW069 on cell viability in parental and DTX-resistant cell lines in DU145 and C4-2 cells. The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01. (**B**) The effect of the combination of DTX and CW069 on apoptosis in parental and DTX-resistant cell lines in DU145 and C4-2 cells. The results are expressed as the mean and S.D. of triplicate measurements. \* *p* < 0.01.

**Table 1.** The combination index (CI) values for the combination of docetaxel (DTX) and CW069 in DTX-resistant cell lines.


CI: combination index; DU145-DR: docetaxel-resistant DU145; C4-2-DR: docetaxel-resistant C4-2; CI = 1: an additive effect; CI < 1: a synergistic effect.
