Validation of Polo-like kinase 1 as a therapeutic target in pancreatic cancer cells

Abstract

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase and plays a critical role in mitosis. PLK1 has also been regarded as a valuable target for cancer treatment, and several PLK1 inhibitors are currently undergoing clinical investigations. In this study, our data show that the expression level of PLK1 is upregulated in human pancreatic cancer cells. Molecular modeling studies indicate that DMTC inhibits PLK1 activity through competitive displacement of ATP from its binding pocket. Our data further show that DMTC suppresses the proliferation of pancreatic cancer cells and induces the formation of multinucleated cells, ultimately resulting in apoptosis. In addition, combination index analysis demonstrates that DMTC acts synergistically with the chemotherapeutic drug gemcitabine in inhibiting the proliferation of pancreatic cancer cells. These results thus suggest a potential of using PLK1 inhibitors for the treatment of pancreatic cancer.

Figure 1. Expression levels of PLK1 mRNA and protein in human pancreatic cancer cells. (A) RT-PCR analysis of PLK1 mRNA expression in normal human pancreatic cells and five pancreatic cancer cell lines. (B) Experiments were performed as in (A) and the relative PLK1 mRNA levels were then quantified using GAPDH as the internal standard. (C) Western blotting analysis of PLK1 protein expression in normal and tumor-derived pancreatic cells. (D) Experiments were performed as in (C) and the relative PLK1 protein levels were then quantified using β-actin as the internal standard. Values, averages of two independent experiments; bars, SD.

Figure 2. Molecular modeling of the interaction between DMTC and PLK1. (A) Schematic models showing PLK1 kinase domain with ADP (left panel) or DMTC (right panel). These models were created by molecular docking as described in Section 2.5. (B) Details of important interactions between DMTC and PLK1. DMTC is color-coded with ivory showing carbon, gray depicting hydrogen, red representing oxygen, blue standing for nitrogen and yellow symbolizing sulfur. Amino acid residues of PLK1 involved in the interaction with DMTC are labeled.

Figure 3. Inhibition of PLK1 activity with DMTC prevents the proliferation of human pancreatic cancer cells. (A) EPP85 cells were treated with varying concentrations of DMTC for 72 h, and the percentage of cell proliferation was measured by SRB-based in vitro cell proliferation assay. The drug concentration needed for 50% inhibition of cell proliferation (IC₅₀) is shown at the upper side. (B) Phase contrast images of EPP85 cells treated with 20 nM DMTC or equal volume of the DMSO control for 0, 24, 48 or 72 h. (C) EPP85 cells were treated for 72 h with 20 nM DMTC, 50 nM DMTC or DMSO as the control. After 2 weeks of culture, the colonies were shown with crystal violet staining. (D) Experiments were performed as in (C), and the number of colonies in each well was then quantified. Values, averages of three independent experiments; bars, SD; **p < 0.01.

Figure 4. DMTC causes the accumulation of cells with abnormal nuclei and triggers apoptosis in pancreatic cancer cells. (A) Immunofluorescence images of microtubules (red) and DNA (blue) in EPP85 cells treated for 24 h with 20 nM DMTC or the DMSO control. (B) Cells were treated with 20 nM DMTC or DMSO (control) for 0, 24 or 48 h, and the percentage of cells with abnormal nuclei was then quantified by immunofluorescence staining of microtubules and DNA as in (A). Values, averages of three independent experiments; bars, SD; **p < 0.01. (C) Quantitation of apoptosis by annexin V/PI staining in EPP85 cells treated for 72 h with 20 nM DMTC or DMSO (control).

Figure 5. Treatment interaction effects of DMTC and gemcitabine in inhibiting EPP85 cell proliferation. (A) Cells were treated with varying concentrations of gemcitabine alone for 72 h, and the percentage of cell proliferation was measured by SRB-based in vitro cell proliferation assay. (B) Cells were treated with varying concentrations of DMTC and gemcitabine in combination at a fixed ratio of 1:10 for 72 h, and the percentage of cell proliferation was measured as in (A). Values, averages of three independent experiments; bars, SD (C) Treatment interaction effects of DMTC and gemcitabine were determined by calculating the CI values for each fraction affected using the CalcuSyn program. CI < 1, synergistic interactions; CI = 1, additive interactions; CI > 1, antagonistic interactions.