Abstract
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 in vitro 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.
Highlights
In recent years, multi-target anticancer drugs have become the focus of cancer therapy
We designed and synthesized a series of α, β-unsaturated ketone derivatives, including HMNE3, which retain the structural characteristics of sunitinib, the basic structure of the α, β-unsaturated ketone of tyrosine kinase inhibitors, and the typical fluoroquinolone structure of topoisomerase inhibitors (Fig 1B)
We have designed and synthesized twenty α, β-unsaturated ketone derivatives, which retain the structural characteristics of sunitinib (Fig 1A), and the typical fluoroquinolone structure of topoisomerase inhibitors (Fig 1B)
Summary
Multi-target anticancer drugs have become the focus of cancer therapy. Tyrosine phosphorylation plays very important roles in regulating cancer cell behavior, including proliferation, motility and differentiation [1,2,3]. Sunitinib (Fig 1A) is an oral, multi-target inhibitor of tyrosine kinases that inhibits the activities of c-Src, Bcr-Abl, and other kinases [6, 7]. It has been approved for clinical use in patients with renal carcinoma, as well as neuroendocrine and breast cancers. A clinical survey indicated that acquired resistance and toxicities are the main side effects, which limit the use of sunitinib in the treatment of other cancers, pancreatic cancer [8, 9]
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