Hypoxia-induced activity loss of a photo-responsive microtubule inhibitor azobenzene combretastatin A4

doi:10.1007/s11705-019-1864-6

Front. Chem. Sci. Eng.

2020, Vol. 14

Issue (5) : 880-888 https://doi.org/10.1007/s11705-019-1864-6

RESEARCH ARTICLE

Hypoxia-induced activity loss of a photo-responsive microtubule inhibitor azobenzene combretastatin A4

Yang An¹, Chao Chen¹, Jundong Zhu¹, Pankaj Dwivedi², Yanjun Zhao¹(

), Zheng Wang¹(

)

¹. School of Pharmaceutical Science & Technology, Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China
². School of Engineering Science,?University of Science and Technology of China, Hefei 230027,?China

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Abstract

The conformation-dependent activity of azobenzene combretastatin A4 (Azo-CA4) provides a unique approach to reduce the side-effects of chemotherapy, due to the light-triggered conformation transition of its azobenzene moiety. Under hypoxic tumor microenvironment, however, the high expression of azoreductase can reduce azobenzene to aniline. It was postulated that the Azo-CA4 might be degraded under hypoxia, resulting in the decrease of its anti-tumor activity. The aim of this study was to verify such hypothesis in HeLa cells in vitro. The quantitative drug concentration analysis shows the ratiometric formation of degradation end-products, confirming the bioreduction of Azo-CA4. The tubulin staining study indicates that Azo-CA4 loses the potency of switching off microtubule dynamics under hypoxia. Furthermore, the cell cycle analysis shows that the ability of Azo-CA4 to induce mitotic arrest is lost at low oxygen content. Therefore, the cytotoxicity of Azo-CA4 is compromised under hypoxia. In contrast, combretastatin A4 as a positive control maintains the potency to inhibit tubulin polymerization and break down the nuclei irrespective of light irradiation and oxygen level. This work highlights the influence of hypoxic tumor microenvironment on the anti-tumor potency of Azo-CA4, which should be considered during the early stage of designing translational Azo-CA4 delivery systems.

Keywords hypoxia microtubule inhibitor drug delivery azo-combretastatin A4 photo-responsive

Corresponding Author(s): Yanjun Zhao,Zheng Wang

Just Accepted Date: 15 November 2019 Online First Date: 19 December 2019 Issue Date: 25 May 2020

Cite this article:

Yang An,Chao Chen,Jundong Zhu, et al. Hypoxia-induced activity loss of a photo-responsive microtubule inhibitor azobenzene combretastatin A4[J]. Front. Chem. Sci. Eng., 2020, 14(5): 880-888.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-019-1864-6
https://academic.hep.com.cn/fcse/EN/Y2020/V14/I5/880

Fig.1 Schematic illustration of conformational transformation and breakdown of Azo-CA4 in the presence of different triggers: (A) proposed degradation products of Azo-CA4 under hypoxia, (B) Azo-CA4 in the hypoxic tumor microenvironment, and (C) light-triggered reversible transition of Azo-CA4 from the inactive “trans” form to the active “cis” form, and hypoxia-triggered breakdown of Azo-CA4 into two end-products.

Fig.2 Viability of HeLa cells in response to different microtubule inhibitors under hypoxia or normoxia (n = 4): (a) CA4 without light treatment, (b) CA4 with light irradiation (365 nm, 10 mW/cm², 10 min, 12 times/day), (c) Azo-CA4 without light treatment, (d) Azo-CA4 with light irradiation, (e) summary of IC₅₀ regarding CA4, and (f) summary of IC₅₀ regarding Azo-CA4. Light irradiation was carried out at 365 nm and 10 mW/cm² for 10 min and 12 times per day; * indicates p<0.05, and n.s. indicates no significance.

Fig.3 Tubulin stained in HeLa cells was treated by fresh cell culture medium (control), free CA4 (30 nmol/L) and Azo-CA4 (60 mmol/L) under normoxia or hypoxia for 24 h. b-Tubulin was stained by Alexa Fluor 488-labelled goat anti-rabbit secondary antibody (green) and nucleus was stained by DAPI (blue). The light irradiation conditions (Laser ON) were 365 nm, 10 mW/cm², 10 min/h, and 12 times/day. Scale bar: 10 mm.

Fig.4 Cell cycle analysis of HeLa cells treated by CA4, Azo-CA4, and only cell culture medium (control) under (a) normoxia and dark (Laser OFF), (b) normoxia and light irradiation (Laser ON), (c) hypoxia and dark, and (d) hypoxia and light irradiation. Light irradiation conditions: 365 nm, 10 mW/cm², 10 min/h, and 12 times/day.

Fig.5 Intracellular quantification of Azo-CA4 and its degradation products (5-amino-2-methoxyphnol and 3,4,5-trimethoxyaniline) under normoxia or hypoxia (n = 4).

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