Abstract
Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 µM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.
Highlights
apurinic endonuclease 1 (APE1) is a multifunctional protein with distinct activities assigned to different parts of its structure
Since past studies had shown that lucanthone inhibits DNA and RNA synthesis [53] [54] with or without affecting protein synthesis [53], we determined the effect of lucanthone on APE1 protein expression in the U251 1–5 APE1 overexpressor cell line pre-treated with 10 mg/ml cycloheximide (CHX) for 4 h
We found that de novo APE1 synthesis was not affected significantly, as seen by the near normal levels of intact APE1 in CHX treated cells (Figure 1)
Summary
APE1 ( termed Ref-1, APEX, HAP1, AP endo) is a multifunctional protein with distinct activities assigned to different parts of its structure. Silber et al showed that APE1 repair activity, which is increased by oxidative stress, contributes to resistance of human glioma cells to alkylating agents [7]. Human glioma cell lines that show lower APE1 expression were more sensitive to methyl methanesulfonate (MMS) and H2O2, known inducers of AP sites and single strand breaks in DNA [8]. When APE1 was over-expressed in U251 cells, they became more radioresistant contingent on the level of APE1 over-expression, whereas siRNA depletion of APE1 was associated with radiation sensitivity This correlation was reiterated by recent studies where APE1 siRNA down-regulation in either colorectal tumor cells in vitro or in a subcutaneous nude mouse colon cancer model enhanced radiosensitivity as revealed by increased apoptosis [11]. In addition to the siRNA studies, we modulated APE1 repair nuclease function using two of its known small molecule inhibitors, lucanthone (1-[2-diethylaminoethylamino]-4-methylthioxanthen-9-one) [12] and CRT0044876 (7Nitroindole-2-carboxylic acid) [13], and showed that APE1 inhibition resulted in increased radiosensitivity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
