Chiral platinum (II)-4-(2,3-dihydroxypropyl)- formamide oxo-aporphine (FOA) complexes promote tumor cells apoptosis by directly targeting G-quadruplex DNA in vitro and in vivo

Abstract

Three platinum(II) complexes, 4 (LC-004), 5 (LC-005), and 6 (LC-006), with the chiral FOA ligands R/S-(±)-FOA (1), R-(+)-FOA (2) and S-(-)-FOA (3), respectively, were synthesized and characterized. As potential anti-tumor agents, these complexes show higher cytotoxicity to BEL-7404 cells than the HL-7702 normal cells. They are potential telomerase inhibitors that target c-myc and human telomeric G-quadruplex DNA. Compared to complexes 4 and 5, 6 exhibited higher binding affinities towards telomeric, c-myc G-quadruplex DNA and caspase-3/9, thereby inducing senescence and apoptosis to a greater extent in tumor cells. Moreover, our in vivo studies showed that complex 6 can effectively inhibit tumor growth in the BEL-7404 and BEL-7402 xenograft mouse models and is less toxic than 5-fluorouracil and cisplatin. The effective inhibition of tumor growth is attributed to its interactions with 53BP1, TRF1, c-myc, TRF2, and hTERT. Thus, complex 6 can serve as a novel lead compound and a potential drug candidate for anticancer chemotherapy.

Keywords: G-quadruplex DNA; antitumor activity; chiral platinum(II) complex; oxoaporphine; telomerase.

Figure 1. The structures of G4-DNA binders and telomerase inhibitors

Figure 2. Complexes 4–6 induced cell senescence

(A) Complexes 1–6 and cisplatin towards six cancer-cell lines and one HL-7702 cell line for 48 h. (B) and (C) Complexes 4–6 (10 μM) treated of BEL-7404 cells for 8.0 h at 37 °C, comparing with cisplatin (10 μM), respectively. Pt content in whole cell (B) and in different fraction (C) were measured by ICP-MS. (D) Complexes 4–6 (2.0 μM) induce cell senescence in BEL-7404 cells: these cells were treatment of complexes 4–6 with 2.0 μM for 7d, and using β-galactosidase stain were examined by fluorescence microscopy (Nikon Te2000 microscope, 200×).

Figure 3. Complexes 4–6 induced apoptosis by triggering caspase-3/9 activity and caused S phase arrest in BEL-7404 cells

(A) Effect of cell apoptosis of BEL-7404 treated with complexes 4–6 (10 μM) for 24 h compared with the untreated cells. (B) Cell cycle effects of BEL-7404 cells treated with complexes 4–6 at 10 μM for 48 h by flow cytometry. (C) and (D) The caspase-3 (C) and caspase-9 (D) protein expression was assessed by flow cytometry following treatment of BEL-7404 cells with complexes 4–6 (10 μM) for 24 h.

Figure 4. Complexes 4–6 induced telomeres damage and inhibited the telomerase activity through directly regulating the mRNA level of c-myc promoter (Pu27)

(A) ΔTm data (°C) of 1.0 μM HTG21, Pu39 and c-myc G4s and duplex DNA (F32T+H20M) treated with complexes 1–6 at 0–2.0 μM were evaluated by RT-PCR. (B) The levels of TRF2, 53BP1, and TRF1 in BEL-7404 cells treated with complexes 4–6 at 10 μM for 24 h were examined by Western blot. (C) The whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against TRF2, 53BP1, and TRF1. The same blots were stripped and re-probed with β-actin antibody to show equal protein loading. Western blotting bands from three independent measurements were quantified with Image J. in (B). (D) The influence of complexes 4–6 (10 μM) on the telomerase activity of the BEL-7404 cells for 24 h. (E) The investigations of the expression of c-myc and hTERT in the BEL-7404 tumor cells when incubated with complexes 4–6 (10 μM) for 24 h. C-myc and hTERT protein levels in BEL-7404 cells were analyzed by western blot. (F) The whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against c-myc and hTERT. The same blots were stripped and re-probed with β-actin antibody to show equal protein loading. Western blotting bands from three independent measurements were quantified with Image J. in (E). (G) qRT-PCR analysis of the expression levels of hTERT and c-myc in the BEL-7404 cells treated with complexes 4–6 (10 μM). The BEL-7404 cells (5×10⁵) were treated with complexes 4–6 (10 μM) for 24 h. The total RNA in the cells was extracted and subjected to reverse transcription, followed by PCR for c-myc, hTERT, and GAPDH (control). (H) and (I) The investigations of the role of the transfections of EGFP plasmid vector (H) and c-myc plasmid vector (I) in the BEL-7404 tumor cells when incubated with complexes 4–6 (10 μM) for 24 h. First, 2.0 μg of EGFP-carrying plasmid vector or 2.0 μg of c-myc-carrying plasmid vector was cotransfected into BEL-7404 cells using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA). Complexes 4–6 (10 μM) were then added, respectively, into medium at 6.0 h after transfection of c-myc plasmid. At another 24 h after treatment with complexes 4–6 (10 μM), these cells were imaged using Nikon TE2000 (Japan) scanning fluorescent microscope or were examined by Multimodel Plate Reader with luciferase reporter gene assay kit.

Figure 5. Complex 6 exhibited antitumor activity in BEL-7404 and BEL-7402 xenograft models

(A–D) In vivo tumor growth inhibition activity of complex 6 (4.0, 8.0 mg/kg/d), and 5-FU (20 mg/kg/2 days) treated with BEL-7404 xenograft model. (E–H) In vivo tumor growth inhibition activity of complex 6 (4.0, 8.0 mg/kg/d) treated of BEL-7402 model with 4.0 and 8.0 mg/kg/d, comparing with cisplatin (2 mg/kg/2 days). (A) and (E). Changes in tumor volume between treatment groups (including complex 6 (4.0, 8.0 mg/kg/d), 5-FU (20 mg/kg/2 days) or cisplatin (2 mg/kg/2 days)) and vehicle (saline) group of BEL-7404 tumor-bearing mice and BEL-7402 tumor-bearing mice. Data of tumor growth were tracked by the mean tumor volume (mm³) ± SD (n = 6) and calculated as percent TGI (tumor growth inhibition, %) values. (B and F) Relative body weight change by considering the body weight at the start of the treated group as 100%, the percent weight loss or gain was calculated on subsequent days of treatment. (C) and (G) Tumor weight between treatment groups and vehicle (saline) group of BEL-7404 tumor-bearing mice and BEL-7402 tumor-bearing mice. (***) P<0.05, (**) P<0.05, p vs vehicle control. (D) and (H) Photographs of harvested tumors from vehicle group and each treatment groups. (I) The expression protein level of TRF1, mutp53, hTERT, TRF2, c-myc, and 53BP1 were analyzed by western blot in BEL-7404 xenograft models treated with 8 mg/kg and 4 mg/kg complex 6, and 20 mg/kg 5-FU, respectively. (J) The whole-BEL-7404 xenograft model extracts were prepared and analyzed by Western blot analysis using antibodies against TRF1, mutp53, hTERT, TRF2, c-myc, and 53BP1. The same blots were stripped and re-probed with β-actin antibody to show equal protein loading. Western blotting bands from three independent measurements were quantified with Image J. in (I).

Figure 6. RT-qPCR array for determining mRNA levels of telomeres/telomerase-related gene expressions in BEL-7404 cells after treatment with complex 6 at 10 μM for 24 h

Figure 7. Proposed antitumor mechanisms for chiral platinum(II) complexes 4–6