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. 2020 Feb 25;11(12):3215-3222.
doi: 10.1039/c9sc05997k.

Combination of chemotherapy and oxidative stress to enhance cancer cell apoptosis

Xinming Li  1 , Yanan Hou  1 , Jintao Zhao  1 , Jin Li  1 , Song Wang  1 , Jianguo Fang  1
Affiliations

Combination of chemotherapy and oxidative stress to enhance cancer cell apoptosis

Xinming Li et al. Chem Sci. .

Abstract

Cancer cells are vulnerable to reactive oxygen species (ROS) due to their abnormal redox environment. Accordingly, combination of chemotherapy and oxidative stress has gained increasing interest for the treatment of cancer. We report a novel seleno-prodrug of gemcitabine (Gem), Se-Gem, and evaluated its activation and biological effects in cancer cells. Se-Gem was prepared by introducing a 1,2-diselenolane (a five-membered cyclic diselenide) moiety into the parent drug Gem via a carbamate linker. Se-Gem is preferably activated by glutathione (GSH) and displays a remarkably higher potency than Gem (up to a 6-fold increase) to a panel of cancer cell lines. The activation of Se-Gem by GSH releases Gem and a seleno-intermediate nearly quantitatively. Unlike the most ignored side products in prodrug activation, the seleno-intermediate further catalyzes a conversion of GSH and oxygen to GSSG (oxidized GSH) and ROS via redox cycling reactions. Thus Se-Gem may be considered as a suicide agent to deplete GSH and works by a combination of chemotherapy and oxidative stress. This is the first case that employs a cyclic diselenide in prodrug design, and the success of Se-Gem as well as its well-defined action mechanism demonstrates that the 1,2-diselenolane moiety may serve as a general scaffold to advance constructing novel therapeutic molecules with improved potency via a combination of chemotherapy and oxidative stress.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Fig. 1
Fig. 1. Structure of the molecules used in this study.
Scheme 1
Scheme 1. Synthesis of target molecules. Reagents and conditions: (a) N2H4·H2O, Se, and 65 °C; (b) N2H4·H2O and KOH; (c) HCl, 0 °C, and air; (d) SOCl2 and MeOH; (e) TsOH, 2,2-dimethoxypropane, and DCM; (f) NaBH4 and MeOH; (g) MSCl, Et3N, and DCM; (h) CH3SO3H, EtOH, and reflux; (i) Na/naphthalene/THF and Se; (j) BnBr, KOH, 2-MeTHF, and TBAHS; (k) MeOH, KOH, and O2; (l) BrBn, KOH, 2-MeTHF, and TBAHS; (m) imidazole, TBDMSCl, and DMF; (n) triphosgene, pyridine, and DCM; (o) TBAF and THF; (p) 1,2-diselenolan-4-ol, pyridine, and toluene.
Fig. 2
Fig. 2. Reduction of different prodrugs by GSH and TrxR/Trx. (A) Reduction of different compounds by GSH. NADPH (200 μM), GSH (5 mM) and GR (0.5 U mL−1) were incubated at 37 °C in TE buffer (50 mM Tris–HCl and 1 mM EDTA, pH 7.4), and the absorbance at 340 nm was monitored. After the mixture was incubated for 5 min, compounds (100 μM) were added. (B) Reduction of S–Gem and Se–Gem by TrxR/Trx. NADPH (200 μM) and different amounts of TrxR with or without Trx were incubated at 37 °C in TE buffer, and the absorbance at 340 nm was monitored. After the mixture was incubated for 5 min, compounds (100 μM) were added. (C) Reduction of Se–Gem by GSH. The reaction conditions were the same as those described in (A) except that the GSH concentrations vary. (D) Aerobic and anaerobic reduction of Se–Gem by GSH. All experiments were performed in triplicate and the representative results are shown.
Fig. 3
Fig. 3. GSH-mediated Gem release from Se–Gem. (A) Se–Gem (100 μM) was incubated with GSH (5 mM) in TE buffer at 37 °C under air conditions, and the reaction mixture was analyzed by HPLC at the indicated time points. Quantification of the time-dependent release of Gem and generation of GSSG is shown in (B) and (C). All experiments were performed in duplicate, and the representative results are shown.
Scheme 2
Scheme 2. (A) Activation of Se–Gem by GSH. (B) Redox cycling reactions of selenolate, oxygen and GSH.
Fig. 4
Fig. 4. Formation of a selenolate intermediate and production of superoxide in the process of GSH-mediated Se–Gem activation. (A) Time-dependent fluorescence increase of the reaction mixture upon incubation with additional GSH (1 mM) and the specific selenolate probe Sel-green (10 μM). The detailed conditions were described in the Experimental section. The inset shows the fold of fluorescence increase (F/F0) as a function of incubation time. (B) Time-dependent increase of fluorescence upon incubation of Sel-green with varying concentrations of the authentic selenocompound SeW. The structure of SeW is shown. The inset shows the linear relationship of the rates of fluorescence increase and the concentrations of SeW. (C) Production of superoxide in the process of GSH-mediated Se–Gem reduction. GSH (100 μM) and ferric cytochrome c (1 mg mL−1) were incubated in TE buffer for 6 min, and then Se–Gem (20 μM) was added. After the mixture was incubated for another 8 min, SOD (150 U) was added. The absorbance spectra of the reaction mixture were recorded every 2 min. (D) Time-dependent change of the absorbance at 550 nm. The experimental conditions were the same as those described in (C), and the concentration of S–Gem is 20 μM. All experiments were performed in triplicate, and the representative results are presented.
Fig. 5
Fig. 5. Induction of oxidative stress by Se–Gem. (A) Alteration of the GSH/GSSG ratio in Hep G2 cells upon treatment with different compounds. The cells were treated with indicated compounds for 72 h, and the intracellular GSH/GSSG ratio was determined. (B) Alteration of total cellular thiols in Hep G2 cells upon treatment with different compounds. The cells were treated with indicated compounds for 72 h, and the cellular total thiols were determined. (C) Accumulation of ROS in Hep G2 cells upon Se–Gem treatment. The cells were treated with different compounds for the indicated times, and the cellular ROS level was determined by DCFH-DA staining. (D) Accumulation of superoxide in Hep G2 cells upon Se–Gem treatment. The cells were treated with different compounds for the indicated times, and the cellular superoxide level was determined by DHE staining. Scale bars = 25 μm. Quantification results of the relative fluorescence intensity (RFI) in individual cells by ImageJ are shown in (E) and (F). All experiments were performed in triplicate. The representative results for (C) and (D) are shown, and others are presented as mean ± SE. In (A) and (B), the control groups were treated without drugs but with the same amount of DMSO (0.1%, v/v). *, P < 0.05 and **, P < 0.01 vs. the control groups, and ## < 0.01 among different groups. In (E) and (F), **, P < 0.01 among different groups.
Fig. 6
Fig. 6. Induction of apoptosis by Se–Gem. (A)–(D) Hep G2 cells were treated with Se–Gem for 48 h, and the apoptotic cell death was evaluated by the Annex V-FITC/PI double staining assay. The control groups were treated without drugs but with the same amount of DMSO (0.1%, v/v). (E) Quantification of live cells, apoptotic cells and necrotic cells from the scatter plots (A)–(D). All experiments were performed in triplicate, and the representative results are shown.
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References

    1. Reczek C. R. Chandel N. S. Curr. Opin. Cell Biol. 2015;33:8–13. doi: 10.1016/j.ceb.2014.09.010. - DOI - PMC - PubMed
    1. Holmstrom K. M. Finkel T. Nat. Rev. Mol. Cell Biol. 2014;15:411–421. doi: 10.1038/nrm3801. - DOI - PubMed
    1. Sies H. Berndt C. Jones D. P. Annu. Rev. Biochem. 2017;86:715–748. doi: 10.1146/annurev-biochem-061516-045037. - DOI - PubMed
    1. Hatem E. El Banna N. Huang M. E. Antioxid. Redox Signaling. 2017;27:1217–1234. doi: 10.1089/ars.2017.7134. - DOI - PubMed
    1. Lu J. Holmgren A. Free Radical Biol. Med. 2014;66:75–87. doi: 10.1016/j.freeradbiomed.2013.07.036. - DOI - PubMed