Design, synthesis and anticancer activity of superoxide anion-releasing beta-galactoside prodrugs
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摘要:
以能在细胞内循环释放超氧阴离子（O2−）的蒽醌类化合物HAQ-OH和AQ-OH为原药，设计并合成了4个全新的蒽醌β-半乳糖苷前药，期望利用Warburg效应和肿瘤细胞内过表达的β-半乳糖苷酶选择性地在肿瘤细胞内释放O2−杀死肿瘤细胞。细胞实验表明，前药Gal-HAQ和Gal-AQ可选择性地抑制β-半乳糖苷酶过表达的卵巢癌OVCAR-3细胞的增殖并诱导细胞凋亡。O2−荧光探针发现，Gal-HAQ和Gal-AQ可时间依赖性地在OVCAR-3细胞内释放O2−，并且O2−对于其抗肿瘤活性至关重要。前药Gal-HAQ和Gal-AQ还可有效清除高表达β-半乳糖苷酶的衰老细胞，而不影响非衰老细胞，进一步证明了前药依赖β-半乳糖苷酶发挥其细胞毒性。综上，本研究发现的能响应β-半乳糖苷酶释放O2−的前药Gal-HAQ和Gal-AQ有望作为新型的抗肿瘤候选分子。
Abstract:Four novel β-galactoside prodrugs were designed and synthesized from anthraquinones HAQ-OH and AQ-OH in an attempt to use the prodrugs to selectively release superoxide anion (O2−) in cancer cells and to achieve selected anticancer activity by utilizing the Warburg effect and the elevated level of β-galactosidase in certain cancer cells. Cellular assays showed that the prodrugs Gal-HAQ and Gal-AQ selectively inhibited the proliferation and induced apoptosis of ovarian cancer OVCAR-3 cells overexpressing β-galactosidase. Using O2− fluorescent probe, it was found that in OVCAR-3 cells Gal-HAQ and Gal-AQ could time-dependently release O2−, which was essential for their anticancer activity. Furthermore, it was found that Gal-HAQ and Gal-AQ were effective senolytics toward senescent cells overexpressing β-galactosidase without affecting the viability of corresponding non-senescent cells, further confirming the β-galactosidase-dependent cytotoxicity of the prodrugs. In conclusion, Gal-HAQ and Gal-AQ, which release O2− in response to β-galactosidase, are expected to serve as candidate prodrugs targeting cancer cells.
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Keywords:
- superoxide anion /
- β-galactoside prodrugs /
- anticancer drugs
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Figure 1. Design of O2−-releasing β-galactoside prodrugs of 5-hydroxy-1,2,3,4,4a,9a-hexahydro-1,4-acetyl-9,10-anthraquinone (HAQ-OH) and 5-hydroxy-1,2,3,4-tetrahydro-1,4-ethylidene-9,10-anthraquinone (AQ-OH)
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1. Synthetic route of target prodrugs Gal-HAQ, Gal-AQ, Gal-HAQ-NO2, and Gal-AQ-NO2
Reagents and conditions: (a) CH2Cl2, HBr, CH3COOH, r.t.; (b) CH2Cl2, H2O, NaOH, 4-hydroxybenzaldehyde or 3-nitro-4-hydroxybenzaldehyde, r.t.; (c) THF, NaBH4, r.t.; (d) CH2Cl2, PBr3, r.t.; (e) CH2Cl2, Ag2O, HAQ-OH or AQ-OH, r.t..
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Figure 2. Stability of prodrugs in PBS as analyzed by HPLC
A:Stability of Gal-HAQ-NO2 in PBS; B:Stability of Gal-AQ-NO2 in PBS
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Figure 3. Deacetylation of prodrugs Gal-HAQ’ (A)and Gal-AQ’(B) in A-549 cells as evidenced by mass spectrometry
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Figure 4. Release of HAQ-OH (A) and AQ-OH (B) from prodrugs in OVCAR-3 cells as evidenced by HPLC
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Figure 5. Determination of intracellular O2−generation by dihydroethidium (DHE) probe
A: Principle of O2− detection by DHE; B: Generation of O2− by HAQ-OH, AQ-OH, Gal-HAQ and Gal-AQ in OVCAR-3 or HEK-293 cells after 12 h;C: Generation of O2− by Gal-HAQ and Gal-AQ in OVCAR-3 cells at different time ($\overline{\text{x}} \text{ ± } \text{s} $, n = 3)
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Figure 6. Effect of 2,2,6,6-tetramethylpiperidoxyl (TEMPOL) (50 μmol/L) on cytotoxicity of prodrugs in OVCAR-3 cells as analyzed by MTT assay ($\overline{\text{x}} \text{ ± } \text{s} $, n = 3)
A: Effect of TEMPOL on cytotoxicity of Gal-HAQ in OVCAR-3(*P < 0.001 vs Gal-HAQ); B:Effect of TEMPOL on cytotoxicity of Gal-AQ in OVCAR-3(*P < 0.001 vs Gal-AQ)
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Figure 7. Death- and apoptosis-inducing effects of Gal-HAQ and Gal-AQ
A： Cell live/death staining of Gal-HAQ and Gal-AQ in OVCAR-3 cells（scale bar: 50 μmol/L）; B：Annexin V-FITC/PI staining of apoptosis cells treated by indicated compounds (20 μmol/L)
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Figure 8. Effects of Gal-HAQ and Gal-AQ on non-senescent/senescent A-549 or L-02 cells
A： Cytotoxicity of Gal-HAQ and Gal-AQ in non-senescent/senescent A-549 or L-02 cells as analyzed by MTT assay ($\overline{\text{x}} \text{ ± } \text{s} $, n = 3). *P < 0.001, **P < 0.01 vs non-senescent A-549 cells; **P < 0.01, ***P < 0.05 vs non-senescent L-02 cells; B： Cytotoxicity of Gal-HAQ and Gal-AQ in non-senescent/senescent A-549 cells as revealed by crystal violet stain
Table 1 Cytotoxicity of indicated compounds in OVCAR-3, HEK-293, and L-02 cells as measured by MTT assay ($\overline{\text{x}} \text{ ± } \text{s} $, n = 3)
Compd. IC50/(μmol/L) OVCAR-3 HEK-293 L-02 HAQ-OH 22.0 ± 0.9 18.0 ± 1.1 9.5 ± 1.9 AQ-OH 37.7 ± 1.3 22.1 ± 0.5 17.9 ± 3.4 Gal-HAQ 23.8 ± 1.1 ˃800 ˃800 Gal-AQ 105.6 ± 1.5 ˃800 ˃800 Gal-HAQ-NO2 116.1 ± 2.3 125.5 ± 1.1 130.8 ± 2.1 Gal-AQ-NO2 175.6 ± 2.7 271.4 ± 3.1 159.8 ± 2.4 
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Table 2 Effects of glucose transporter-1 (GLUT1) inhibitor phlorizin (1 mmol/L) on the cytotoxicity of indicated compounds in OVCAR-3 cells ($\overline{\text{x}} \text{ ± } \text{s} $, n = 3)
Compd. IC50/(μmol/L) Without phlorizin Phlorizin pretreatment HAQ-OH 23.2 ± 0.5 25.3 ± 1.2 AQ-OH 19.5 ± 0.8 24.8 ± 1.0 Gal-HAQ 23.9 ± 1.4 86.7 ± 2.1* Gal-AQ 105.6 ± 2.0 168.9 ± 2.6* *P < 0.001 vs without phlorizin
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