br Conclusions br Despite cisplatin has been
Despite cisplatin has been acting as the first-line drug for the treatment of NSCLC, it is severely limited by side effects and drug re-sistance. In this study, complex 1 as a Pt(IV) prodrug of cisplatin con-taining NBDHEX (a GSTs inhibitor) was deliberately designed, prepared and studied upon an idea to overcome cisplatin resistance and reduce its toxicity by inhibiting GSTs overexpressed in most of cancer cells. Complex 1 could be reduced to release its active Pt(II) species and axial ligand under the reduction of ascorbic acid. Its cytotoxicity against A549 cells was stronger than that of cisplatin, especially toward cis-platin-resistant A549/DDP cells with a resistance factor value of 0.37. By inhibiting GSTs, the promotion of platinum uptake and DNA plati-nation, more severe DNA damage were observed in cancer cells treated with complex 1, resulting in the reversal of cisplatin-resistance in lung cancer cells. Complex 1 could trigger cell death via an apoptotic pathway and arrested the cell cycle of sensitive A549 cells at G2/M phase, while mainly arrested the cell cycle of resistant A549/DDP cells at S phase. Different from cisplatin, complex 1 could down-regulate Bcl-2 and upregulate cleaved PARP in the cisplatin resistant cells. In vivo tests on A549 xenograft tumor mice model showed that complex 1 not only had the highest tumor inhibiting rate compared with cisplatin, NBDHEX and their physical mixture, but also exhibited least toxic
Fig. 9. Western blot analysis of the mitochondrial related apoptotic proteins and PARP which is associated with DNA repair. Cells were untreated or treated with cisplatin, NBDHEX or complex 1 at a concentration of 20 μM for 12 h.
Fig. 10. (A) Tumor volume in each group during the observation period; (B) Tumor volume inhibitory rate in different treatment groups; (C) mice body weight in each group during the observation period.
among the testes samples. In short, complex 1 owning the GSTs in-hibitory ability presented potent ability for the treatment of NSCLC, which has potential to be developed as an anticancer agent.
4. Materials and methods
4.1. Materials and instrument
All chemicals and solvents were of analytical reagent grade and used without further purification, unless noted specifically. Cisplatin was synthesized in our lab. All gamma-Glu-Cys used in this study were purchased from Cell Signaling Technology (CST). Mass spectra were measured by an Agilent 6224 ESI/TOF MS instrument. 1H NMR and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 on a Bruker 300 MHz or 500 MHz spectrometer. 195Pt NMR spectra were measured in DMSO-d6 with a Bruker 600 MHz spectrometer. Elemental analyses of C, H, and N used a Vario MICRO CHNOS elemental analyzer (Elementar). Waters 1525 HPLC equipment was used to study the purity, stability Journal of Inorganic Biochemistry 193 (2019) 133–142
and releasing ability of the compound. Platinum contents were de-termined by Inductively Coupled Plasma-Mass Spectrometer (ICP-MS, Optima 5300DV, PerkinElmer, USA). Cell cycle and apoptosis experi-ments were measured by flow cytometry (FAC Scan, Becton Dickenson) and analyzed by Cell Quest software.
4.3. Synthesis of oxoplatin
Hydrogen peroxide (30 wt%, 30 mL) was added drop wise to a round bottom flask containing cisplatin (500.0 mg, 1.67 mmol). The reaction mixture was heated to 60 °C for 12 h. The bright yellow solu-tion was kept at room temperature in the dark for overnight to allow crystallization of the product. Yellow crystals were separated by fil-tration, washed with cold water, and dried to get 485.0 mg of oxoplatin, yield 87%.
4.5. Cell lines and cell culture
Fig. 11. H&E staining of the tumor and primary tissues in different treatment group.
4.6. Cell sensitivity to compounds
In vitro anticancer activity of complex 1 was evaluated by MTT as-says, using cisplatin, carboplatin, oxaliplatin, NBDHEX and the physical mixture of cisplatin and NBDHEX as the positive drugs. Cells were grown in 96 well plates at 6000 cells per well in a final volume of 200 μL of culture medium and cultured in an incubator (5% CO2, 37 °C) until the cells reached 70–80% confluency. Samples were dissolved by DMF and diluted with medium to various concentrations (the final concentration of DMF was less than 0.4%). After being incubated at 37 °C for 72 h, cells were stained with MTT (5 mg/mL) for another 4 h and dissolved with 150 μL of DMSO. The UV absorption intensity was detected with an ELISA reader at 490 nm. IC50 values were calculated by SPSS software after three parallel experiments and all experiments were performed in triplicate.