Research paper5-Bromo-oxoisoaporphine platinum(II) complexes exhibit tumor cell cytotoxcicity via inhibition of telomerase activity and disruption of c-myc G-quadruplex DNA and mitochondrial functions
Graphical abstract
Introduction
The c-myc gene encodes a central regulator that regulates the expression of 15% of the genes, including those involved in cell growth, cellular proliferation, apoptosis, and senescence [[1], [2], [3], [4]]. The over-expression of c-myc gene and its protein product is associated with the tumourigenesis of malignant cancers [[1], [2], [3], [4]], which is known to be a key activator for hTERT (human telomerase reverse transcriptase) gene transcription. The NHE III1 (nuclear hypersensitivity element III1) is a guanine-rich 27 base-pair sequence located upstream the P1 promoter of c-myc gene, which controls 80%–90% of the c-myc gene transcription level [[5], [6], [7], [8], [9], [10]]. A variety of compounds or G4s ligands have been reported to induce and stabilize the c-myc NHE III1 G4 DNA and inhibit c-myc gene transcription level, such as quindoline derivatives [[11], [12], [13]], cationic porphyrins [[14], [15], [16], [17]], and platinum complexes [[18], [19], [20], [21], [22], [23], [24], [25]], and ruthenium complexes [[26], [27], [28]], etc. Most of these compounds or complexes comprise with a planar aromatic moiety [3,8,11,[29], [30], [31], [32], [33], [34]], such as phenolic compounds [35], pentacyclic acridine [36], anthracycline [37], ethidium derivatives [38], naphthalene diimide derivatives [39], berberine alkaloid [40], quindoline derivative (SYUIQ-05) [8,12], benzo[c]phenanthridine derivative [30], trinuclear platinum complexes [Pt3Cl3(hptab)][ClO4]3 (hptab = N,N,N′,N′,N″,N″-hexakis(2-pyr-idylmethyl)-1,3,5-tris(aminomethyl)-benzene), [Pt2Cl2(m-tpxa)]Cl2 (m-tpxa = N,N,N′,N′-tetra(2-pyridylmethyl)-m-xylylene diamine) [41], metal-terpyridine [42] and supramolecular Pt(II) complexes [34,43], which significantly improve participating in covalently coordinated, groove binding and loop-binding, π−π stacking and electrostatic interactions with the G-quartet at the end of G4 DNA [3,8,11,[29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]]. Moreover, the extended planar chromophores can significantly improve the biological behavior by G4-ligands, which are similar in size and shape to that of a G-quartet stack on the surface of this structure by π−π interactions or covalently coordinated [3,8,11,[29], [30], [31], [32], [33], [34]]. For example, the cationic porphyrins TMPyP4 [44], the expanded porphyrin Se2SAP [45], and telomestatin have a macrocycle chromophore with a size similar to that of the G-tetrad [3,8,11].
In addition, we have recently designed, synthesized and identified oxoaporphine and oxoisoaporphine metal complexes-based c-myc G4 binder [[24], [25], [26], [46]]. Our previous studies have demonstrated that oxoisoaporphine platinum(II) and Co(II/III) complexes are covalently coordinated to guanine of c-myc G4-DNA [[24], [25], [26], [46]]. Furthermore, a variety of studies have demonstrated that expanding the size of aromatic planar surface of G4-ligands and introduction of key substitutions (eg. halogen, CH3O–, –OH and NO2 substitution, etc.) in these ligands might enhance their binding potency and selectivity on G4 DNA over duplex DNA, thereby leading to higher antitumor activities [8,[47], [48], [49], [50]].
In this study, we designed and synthesized 5-bromo-oxoisoaporphine (H-L) [46] and its two new platinum(II) complexes, [Pt(L)(DMSO)Cl] (1) and [Pt(L)(pn)]Cl (2). In addition, 5-bromo-oxoisoaporphine platinum(II) complexes 1 and 2 contain halogen substitution in H-L ligand or/and different second ligands (pn or Cl and DMSO), which are different from the previously reported oxoisoaporphine platinum(II) Pt1 and Pt2 [24] and 9-amino-oxoisoaporphine platinum(II) complex [25]. The interactions of the two new Pt(II) complexes 1 and 2 with c-myc gene and G4 DNA were examined by RT-PCR, Western blotting, TRAP assay and transfection of Hep-G2 cells with EGFP and c-myc gene vector assays. Results indicated that the inhibition of telomerase by 5-bromo-oxoisoaporphine platinum(II) complexes 1 (10 μM) and 2 (5 μM) was through direct down-regulation of the c-myc/Pu27 G4 in Hep-G2 cells. Furthermore, cytotoxic mechanism studies demonstrated that complexes 1 (10 μM) and 2 (5 μM) triggered cancer cell apoptosis through disruption of the mitochondrial functions.
Section snippets
Synthesis and characterization
5-Bromo-oxoisoaporphine (H-L) was prepared with 4-bromo phenylethylamine and phthalic anhydride using a procedure reported previously [46]. A new square planar platinum(II) complex [Pt(L)(DMSO)Cl] (1) containing substituted 5-bromo-oxoisoaporphine ligand, was obtained by the reaction of the cis-[PtCl2(DMSO)2] with 5-bromo-oxoisoaporphine (H-L). As shown in Scheme 1, the reactions of [Pt(L)(DMSO)Cl] (1) with 1,3-propanediamine (pn) in the presence of methanol generated [Pt(L)(pn)]Cl (2). All the
Conclusion
In summary, [Pt(L)(DMSO)Cl] (1) and [Pt(L)(pn)]Cl (2) of 5-bromo-oxoisoaporphine (H-L) were synthesized and fully characterized. The IC50 values were in the range of 5.06–31.09 μM, in which Hep-G2 cells showed the highest sensitivity to complexes 1 and 2, comparing with the human normal cells (HL-7702, WI-38 and L-o2 cell lines). Their in vitro cytotoxicities were mainly related to their second ligand pn or Cl and DMSO, whereas the pn-containing complex showed the most potent activity in all
Experimental methods
The purity of 5-bromo-oxoisoaporphine platinum(II) complexes 1 and 2 used in the biophysical and biological studies was ≥95%. Both 5-bromo-oxoisoaporphine platinum(II) complexes 1 and 2 were routinely analyzed by HPLC.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (Grants 81473102, 21431001), IRT_16R15, CMEMR2012-A22, the Startup Program of Yulin Normal University (No. G2017009) and Natural Science Foundation of Guangxi Province (Grant2012GXNSFDA053005) as well as “BAGUI Scholar” program of Guangxi Province of China.
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These authors contributed equally to this work.