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  • br Cellular uptake and DNA platination br It

    2019-09-24


    2.5. Cellular uptake and DNA platination
    It has been demonstrated that the reduced intracellular platinum accumulation is an important factor of cisplatin-resistance [6]. So the cellular uptake and DNA platination of cisplatin and complex 1 at dif-ferent concentrations in A549 and A549/DDP Trizma maleate were analyzed by ICP-MS. As shown in Fig. 5 and Table S1, in cisplatin-sensitive A549 cells, the cellular uptake of cisplatin and complex 1 was dose-depen-dent, and the platinum accumulation of complex 1 was 6.3 fold greater than that of cisplatin at a concentration of 10 μM and 2.6 fold at a concentration of 20 μM. Meanwhile, the DNA platination caused by complex 1 was 11.9 fold greater than that of cisplatin at a concentration of 10 μM and 5.1 fold at a concentration of 20 μM. In cisplatin-resistant
    Fig. 5. (A) Cellular uptake and (B) DNA platination of cisplatin and complex 1 at different concentrations in cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells after 12 h of incubation. Results are expressed as the mean ± SD for three independent experiments.
    A549/DDP cells, the uptake of cisplatin was dramatically decreased, but the uptake of complex 1 was still higher than that of cisplatin, which was 26.6 fold as much as that of cisplatin at 10 μM and 8.7 fold at Trizma maleate 20 μM. Besides, the DNA platination in A549/DDP cells caused by cis-platin at 10 μM was not detected, but it was significantly increased under the treatment of complex 1. The enhanced lipophilicity (log P values showed in Table S2) was probably one of the reasons for the increase of cellular uptake, while the dramatic increase of DNA plati-nation was likely to owe to the inhibition of GSTs. The results exhibited that complex 1 owns the capacity of overcoming cisplatin-resistance, the enhanced platinum accumulation and DNA platination may be one of the determinants of platinum sensitivity. Obviously, introduction of a GSTs inhibitor to the Pt(IV) prodrug can significantly promote the ac-cumulation of platinum and DNA platination, leading to the reversal of cisplatin-resistance of lung cancer cells.
    Anticancer effects of platinum-based drugs are proved to be medi-ated by irreversible DNA damage. Comet assay, a gel electro-phoresis–based method that can be used to measure DNA damage in individual eukaryotic cells, is versatile and relatively simple to perform [34]. If the negatively charged DNA was damaged, DNA supercoils were relaxed and broken ends were able to migrate to the anode. The comet head contains the high-molecular-weight DNA and the comet tail con-tains the leading ends of migrating fragments. As presented in Fig. 6, NBDHEX cannot cause DNA damage because its target is not DNA. Cisplatin could trigger distinct DNA damage in sensitive A549 cells as the DNA tails were found, while it was invalid in cisplatin-resistant cells. In contrast, both sensitive and resistant cells treated with complex 1 owned longer length of DNA tails, and the tails in A549/DDP cells were even longer than those of A549 cells, indicating that complex 1 caused more severe DNA damage especially in cisplatin-resistant cells
    Fig. 6. Representative images of nuclei in comet assay. A549 and A549/DDP cells were treated with 20 μM of cisplatin or complex 1 for 12 h.
    due to the enhancement of platinum accumulation. This result is con-sistent with the data obtained by MTT assay and DNA platination test.
    2.7. Apoptosis study
    Apoptotic analysis of complex 1 against A549 and A549/DDP cells was carried out by flow cytometric assay, cisplatin and NBDHEX were used as positive compounds. As shown in Figs. 7 and S6, few apoptotic or necrotic cells were present in both control panels; cisplatin achieved an apoptosis rate of 17.6% (5.1% early apoptosis, 12.5% late apoptosis) with a necrosis rate of 9.4% in sensitive A549 cells, while it was for-celess in resistant A549/DDP cells (4.2% early apoptosis, 5.2% late apoptosis, 0.4% necrosis); however, the apoptosis rate under the treatment with complex 1 at the same concentration strongly increased to 76.5% (24.5% early apoptosis, 52.0% late apoptosis) in sensitive A549 cells with a necrosis rate of 3.2%, and 78.9% (29.5% early apoptosis, 49.4% late apoptosis) in resistant A549/DDP cells with a necrosis rate of 8.9%. It was noted that NBDHEX killed more A549 and A549/DDP cells than complex 1 mainly in the late stage of apoptosis, which may be related to the rapid response of cells toward NBDHEX. All the above results revealed that complex 1 could trigger lung cancer cell