KSP inhibitor ARRY-520 as a substitute for Paclitaxel in Type I ovarian cancer cells

Abstract

Background: We previously described a sub-population of epithelial ovarian cancer (EOC) cells with a functional TLR-4/MyD88/NF-kappaB pathway (Type I EOC cells), which confers the capacity to respond to Paclitaxel, a known TLR-4 ligand, by enhancing NF-kappaB activity and upregulating cytokine secretion - events that are known to promote tumor progression. It is therefore important to distinguish those patients that should not receive Paclitaxel; it is also important to identify alternative chemotherapy options that would benefit this sub-group of patients. The objective of this study is to determine if the KSP inhibitor, ARRY-520, can be a substitute for Paclitaxel in patients with Type I EOC.

Methods: EOC cells isolated from either ascites or tumor tissue were treated with increasing concentrations of ARRY-520 or Paclitaxel and cell viability determined. Activation of the apoptotic pathway was determined using Western blot analysis. Mitochondrial integrity was quantified using JC1 dye. Cytokine profiling was performed from supernatants using xMAP technology. NF-kappaB activity was measured using a Luciferase reporter system. In vivo activity was determined using a subcutaneous xenograft mouse model.

Results: ARRY-520 and Paclitaxel exhibited the same cytotoxic effect on Type I and II cells. The GI50 at 48 h for Type II EOC cells was 0.0015 microM and 0.2 microM for ARRY-520 and Paclitaxel, respectively. For Type I EOC cells, the GI50 at 48 h was > 3 microM and >20 microM for ARRY-520 and Paclitaxel, respectively. Decrease in the number of viable cells was accompanied by mitochondrial depolarization and caspase activation. Unlike Paclitaxel, ARRY-520 did not induce NF-kappaB activation, did not enhance cytokine secretion, nor induce ERK phosphorylation in Type I EOC cells.

Conclusion: Administration of Paclitaxel to patients with high percentage Type I cancer cells could have detrimental effects due to Paclitaxel-induced enhancement of NF-kappaB and ERK activities, and cytokine production (e.g. IL-6), which promote chemoresistance and tumor progression. ARRY-520 has similar anti-tumor activity in EOC cells as that of Paclitaxel. However, unlike Paclitaxel, it does not induce these pro-tumor effects in Type I cells. Therefore, the KSP inhibitor ARRY-520 may represent an alternative to Paclitaxel in this subgroup of EOC patients.

Figure 1

ARRY-520 significantly decreases the number of viable Type II EOC cells. The viability (in percentage, normalized to untreated cells) of EOC cells after treatment with increasing concentrations of ARRY-520 for (a) 24 and (b) 48 hours. Data were compiled from at least three independent experiments, each done in triplicate. Type I cells – R182, 01–19b, R1140; Type II cells – A2780, CP70, 01–28; dotted line corresponds to 50% viability.

Figure 2

ARRY-520 induces apoptosis in Type II EOC cells. Type II EOC cells were treated with 3 μM ARRY-520 for 6, 12, and 24 hours. "0" designation represents non-treated controls. (a) Activity of capase-3, -8, and -9 was measured using Caspase-Glo assay, and (b) effect on XIAP, Caspase-2, and Bid was determined using Western blot analysis. Results shown are for CP70. Similar results were observed with other cells tested.

Figure 3

ARRY-520 induces apoptosis independent of the mitochondrial pathway. (a) Type II EOC cells were treated with 3 μM ARRY-520 for 12 and 24 hours, stained with JC-1 dye as described in the Materials and Methods section, and mitochondrial integrity was analyzed using Flow cytometry. (b) Graphical representation of the percentage of polarized and depolarized cells. Note that ARRY-520 does not induce mitochondrial depolarization. Results shown are obtained with CP70 cells. Similar results were observed with other cells tested.

Figure 4

Differential effect of ARRY-520 and Paclitaxel on NF-κB activation in Type I EOC cells. Cells were transfected with a luciferase reporter plasmid activated by NF-κB and treated with either 3 μM ARRY-520 or 2 μM Paclitaxel. NF-κB activity was measured as luminescence. Data shown are for R182 cells. Similar results were obtained with other Type I EOC cells tested.

Figure 5

Differential effect of ARRY-520 and Paclitaxel on cytokine profile in Type I EOC cells. Cells were treated with ARRY-520 (0.03, 0.3, 3 μM) or Paclitaxel for (0.2, 2, 20 μM) for 48 hrs and levels of secreted cytokines/chemokines were determined using xMAP technology.

Figure 6

Differential effect of ARRY-520 and Paclitaxel on ERK activation in Type I EOC cells. Cells were treated with ARRY-520 (0.03, 0.3, 3 μM) or Paclitaxel for (0.2, 2, 20 μM) for 24 hrs and levels of phospho-ERK (p-ERK) and total ERK (t-ERK) weredetermined by Western blotting.

Figure 7

In vivo activity of ARRY-520 and Paclitaxel. EOC tumors were established s.c. in female nude mice and treatments were given as described in the Materials and Methods section. Tumor size was determined by caliper measurements. (a) A2780 xenograft model and (b) tumors established from a primary culture of EOC cells.