Dual Inhibition of Topoisomerase II and Tyrosine Kinases by the Novel Bis-Fluoroquinolone Chalcone-Like Derivative HMNE3 in Human Pancreatic Cancer Cells

<div><p>Both tyrosine kinase and topoisomerase II (TopII) are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK) detection kit using a horseradish peroxidase (HRP)-conjugated phosphotyrosine (pY20) antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P<0.05), and this effect was accompanied by a decrease in tyrosine kinase activity. HMNE3 potentially inhibited tyrosine kinase activity <i>in vitro</i> with an IC₅₀ value of 0.64±0.34 μmol/L in Capan-1 cells and 3.1±0.86 μmol/L in Panc-1 cells. The activity of c-Src was significantly inhibited by HMNE3 in a dose- and time-dependent manner in different cellular contexts. Compared with the control group, HMNE3 induced increased expression of cellular apoptosis-related proteins. Consistent with cellular apoptosis data, a significant decrease in topoisomerase IIβ activity was noted following treatment with HMNE3 for 24 h. Our data suggest that HMNE3 induced apoptosis in Capan-1 and Panc-1 cells by inhibiting the activity of both tyrosine kinases and topoisomerase II.</p></div

Effects of HMNE3 on cancer cell proliferation.

<p>The logarithmically growing cell lines (T24, Panc-1, BGC-823, PU145, Capan-1, HGC-27, and normal hepatocyte C3A) were treated with various concentrations of HMNE3 for 24, 48 and 72 h. The inhibition of cell proliferation was assessed with the MTT assay, and the data were presented as the means of at least three independent experiments.</p

HMNE3 inhibited tyrosine kinase activity in the Capan-1 (A) and Panc-1 cells (B).

<p>Ninety-six-well plates were pre-coated with 20 μg/mL of a 4:1 poly (Glu,Tyr) solution as a substrate. An ATP solution was added to each well. Varying doses of HMNE3 were added to each test well. The kinase reaction was initiated by the addition of purified tyrosine kinase proteins diluted in 49 μL of kinase reaction buffer. After incubation for 1 h at 37°C, the plate was washed thrice with PBST. An anti-phosphotyrosine (PY99) antibody was then added. After a 30 min incubation at 37°C, the plate was washed thrice, and 100 μL of a horseradish peroxidase-conjugated goat anti-mouse IgG was added. The reaction was terminated by the addition of 50 μL of 2 mol/L H₂SO₄, and the plate was read at 490 nm using a multi-well spectrophotometer.</p

<i>In vitro</i> Topo II activity assay using agarose gel electrophoresis and western blotting.

<p>(A) Approximately 0.25 μg pBR322 DNA was incubated with 1 unit of human Top IIα and 1.6 μM HMNE3 for 30 min at 37°C in a total of 20 μL of reaction buffer. The reaction was then stopped with 2 μL of 10% SDS and 1 μL proteinase K. The samples were subjected to electrophoresis on a 0.8% agarose gel in 1× TAE at 5 V/cm for 1 h. Then, the gel was stained with 0.5 μg/mL of ethidium bromide (EB) for 30 min, destained with distilled water for 30 min, and photographed under a UV trans-illuminator. (B) The expression levels of Top IIα and Top IIβ were detected by western blotting.</p

The structure and name of the bis-fluoroquinolone chalcone-like derivative HMNE3.

<p>(1-cyclopropyl-3-[1-cyclopropyl-6-fluoro-7-piperazin-1-yl-2,3-dihydro quinolin-4(1H)-one-3-ylidenemethyl]-6-fluoro-7-(4-methylpiperazin-1-yl)-quinolin-4 (1H)-one).</p

Role of HMNE3 in the cell cycle progression of Capan-1 cells.

<p>Logarithmically growing Capan-1 cells were treated with 1.6 μM or 3.2 μM HMNE3 for 24 h. Cell cycle was analyzed by flow cytometry. The statistics of three independent experiments are presented. *, P<0.05 compared with the control group; Student’s t test.</p

The IC₅₀ values of HMNE3 in six cell lines following treatment for 48 h.

<p>The IC₅₀ values of HMNE3 in six cell lines following treatment for 48 h.</p

Nuclear staining of Capan-1 cells following 48 h treatment with HMNE3.

<p>Approximately 5×10³ cells/mL were seeded on 35-mm glass slides. After treatment with HMNE3 for 48 h, the cells were washed twice with PBS and incubated with 5 μg/mL Hoechst 33258 for 10 min at 37°C in the dark. The nuclear morphology was then examined under a fluorescent microscope.</p

Inhibition of decatenation activity of topoisomerase II by HMNE3.

<p>All the cells were harvested on ice and nuclear extracts were separated from whole cellular proteins. Total 0.1 μg kDNA and 1unit of human topo IIa were mixed with the reaction buffer at 37°C for 15 min, the reaction was stopped by the addition of 10% SDS. Then, the DNA samples were subjected to electrophoresis in a 1% agarose gel containing 1 ug/ml ethidium bromide.</p

Immunocytochemical staining of activated Caspase-3 in Capan-1 cells following HMNE3 treatment.

<p>Approximately 2×10⁴ cells were plated on coverslips that were pre-placed in 12-well plates. All of the cells were treated with the indicated doses of HMNE3 for 48 h. The cells were fixed and incubated with 5% BSA for 30 min. The cells were incubated with a rabbit anti-human cleaved Caspase-3 antibody overnight at 4°C, washed with PBS, and incubated with a biotinylated horse anti-rabbit IgG antibody for 30 min. After rinsing with PBS for 5 min, streptavidin-horseradish peroxidase enzyme complex was diluted in PBS and incubated with the cells for 30 min. After washing with PBS, the staining was visualized by incubating the cells in DAB and counter-stained with a hematoxylin solution. The cells were imaged using a microscope. * P<0.05 compared with the control group; Student’s t test.</p