Dual Stimuli-Activatable Oxidative Stress Amplifying Agent as a Hybrid

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Dual stimuli-activatable oxidative stress amplifying agent as a hybrid anticancer prodrug Eunji Han, Byeongsu Kwon, Donghyuck Yoo, Changsun Kang, Gilson Khang, and Dongwon Lee Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00683 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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Bioconjugate Chemistry

Dual Stimuli-Activatable Oxidative Stress Amplifying Agent as a Hybrid Anticancer Prodrug Eunji Han1, Byeongsu Kwon1, Donghyuck Yoo1, Changsun Kang1, Gilson Khang1,2 & Dongwon Lee1,2,*

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Department of BIN Convergence Technology, Chonbuk National University, Baekjedaero 567,

Jeonju, Chonbuk, 567-756, Republic of Korea 2

Department of Polymer⋅Nano Science and Technology, Chonbuk National University,

Baekjedaero 567, Jeonju, Chonbuk, 567-756, Republic of Korea

Corresponding Author Dongwon Lee, Phone: +82-63-270-2344. Email: [email protected]. Fax: +82-63-270-2341

ACS Paragon Plus Environment

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ABSTRACT

Compared to normal cells, cancer cells have a higher level of reactive oxygen species (ROS) due to aberrant metabolism and disruption of redox homeostasis which drive their proliferation and promote progression and metastasis of cancers.

The altered redox balance and biological

difference between normal cells and cancer cells provide a basis for the development of anticancer agents which are able to generate pharmacological ROS insults to kill cancer cells preferentially. In this study, we report a new hybrid anticancer drug, termed OSamp, which undergoes esterase- and acid-catalyzed hydrolysis to deplete antioxidant glutathione (GSH) and generate ROS, simultaneously. OSamp significantly elevated oxidative stress in cancer cells, leading to enhanced apoptotic cancer cell death through mitochondrial membrane disruption, cytochrome c release, activation of pro-caspase 3 and deactivation of STAT3 (signal transducer and activator of transcription-3). OSamp intravenously administrated significantly suppressed the tumor growth in a mouse model of tumor xenografts without notable side effects. Oxidative stress amplifying OSamp holds tremendous potential as a new anticancer therapeutics and provides a new therapeutic paradigm which can be extended to development of hybrid anticancer drugs.


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INTRODUCTION ROS (reactive oxygen species) are normal byproducts of various cellular processes, including superoxide anion, hydrogen peroxide (H2O2) and hydroxyl radical and play an important role as a secondary messenger in cellular signaling to activate proliferation and survival pathways.1 Despite their essential roles, overproduction of ROS causes oxidative stress, damaging DNA and promoting mutagenesis in cells.2 Cells that fail to keep the redox balance are prone to oxidative stress-induced cell death. Therefore, cells upregulate multiple antioxidant systems to control the ROS level and maintain ROS homeostasis.1 Under physiological conditions, cells have a basal level of ROS and maintain redox balance through the generation and elimination of ROS. However, compared to normal cells, cancer cells are under oxidative stress with an elevated level of ROS associated with active metabolism and oncogenic transformation.1,3 The increased level of ROS plays critical roles in the growth and migration of cancer cells, maintenance of cancer phenotype and tumor development.4 Cancer cells are also equipped with profoundly altered antioxidant systems to adapt ROS stress to ensure their survival, including glutathione (GSH) which is one of the most abundant and major reducing systems of cells to maintain redox balance.1,5-6 On the basis of the biochemical alteration in cancer cells, there has been increasing interest in the development of strategies to kill cancer cells specifically by elevating oxidative stress.7-8 Increased ROS in cancer cells have been viewed as an adverse and unfavorable event in anticancer therapy due to cancer-promoting effects.2-3 However, a number of recent studies reported that cancer cells under oxidative stress are highly dependent on antioxidant defense systems to counteract damaging effects of ROS and therefore are more susceptible to oxidative insults by exogenous agents which abrogate antioxidant systems or generate further ROS.9-10 A


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straightforward approach to the elevation of oxidative stress is to utilize exogenous ROSgenerating agents such as As2O3, elesclomol and cinnamaldehyde.3,11 Cinnamaldehyde is a major active ingredient in cinnamon, widely used in food and beverage and has been known to exert anticancer and antimicrobial activity through ROS generation.12-13 Inhibition of antioxidant systems is an alternative approach to kill cancer cells preferentially by elevating oxidative stress.8,14-16 GSH is a ubiquitously expressed antioxidant and its essential functions include squelching free radicals and maintaining the redox balance in cells.3,15 Considerable evidence suggests that the GSH depletion by L-buthionine-sulfoximine (BSO) and β-phenylethyl isocyanate elevates oxidative stress, leading to cancer cell death.2-3,17 Anticancer activity of nitric oxide-donating aspirin is also attributed to GSH depletion by quinine methide (QM), which is an intermediate generated from esterase-catalyzed hydrolysis.18 It was also reported that GSH depletion sensitizes cancer cells to ROS-generating agents and synergizes with As2O3 in the induction of apoptosis.15,17

It has been therefore suggested that the

combination of agents with complementary mechanisms of actions could provide an improved therapeutic index while minimizing the detrimental effects on normal cells.8,19-20 In this context, we developed a novel oxidative stress amplifying hybrid anticancer drug (OSamp), which is able to not only deplete GSH but also generate ROS. OSamp was rationally designed to produce two complementary pharmacophores, GSH-depleting QM via esterasecatalyzed hydrolysis and ROS-generating cinnamaldehyde via acid-triggered hydrolysis (Figure 1). The hypothesis in the development of OSamp is that QM-mediated GSH depletion sensitizes cancer cells to cinnamaldehyde-induced ROS insults, leading to preferential cancer cell death. Herein, we report the therapeutic potential of OSamp as a dual stimuli-activatable oxidative stress amplifying anticancer drug using cell cultures and xenograft mouse models.


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Figure 1. Dual stimuli-activatable oxidative stress amplifying hybrid anticancer prodrug, OSamp.

RESULTS Design and synthesis of OSamp OSamp was developed to generate two complementary pharmacophores, ROS-generating cinnamaldehyde and antioxidant-depleting QM, in a dual stimuli-triggered manner.

The

chemical structure and synthetic route of OSamp are shown in Figure 2. Cinnamaldehyde derivative 2 was synthesized from the reaction of cinnamaldehyde and 2-(hydroxymethyl)-2methylpropoane-1,3-diol, followed by the reaction with 1,1-dicarbonyldiimidazole.

1,4-

Hydroxybenzyl alcohol was reacted with benzoyl chloride to synthesize an ester compound 3. OSamp was obtained as white power (∼45% yield) from the reaction of 2 and 3. As shown in Figure 2, cinnamaldehyde-releasing moiety and QM-generating moiety were coupled through a carbonate linkage, which could serve as a good leaving group to allow QM formation. Therefore, OSamp satisfies the requirement for QM generation, that an aromatic ring is substituted with both a masked phenolic group and a para-methylene group attached to a good


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leaving group.18 OSamp was characterized by NMR spectroscopy (Figure S1-2, Supporting Information).

Figure 2. A synthetic route of dual stimuli-responsive OSamp.

Responsiveness of OSamp to acidic pH and esterase OSamp was designed to be activated by acidic pH and esterase to exert anticancer activity by releasing ROS-generating cinnamaldehyde and GSH-scavenging QM, respectively. As shown in Figure 3A, OSamp is expected to undergo esterase-triggered carboxylic ester hydrolysis to generate phenolate, followed by rapid and energetically favorable 1,6-elimination of the leaving group, thereby generating QM. We first investigated the responsiveness of OSamp to esterase by measuring the level of GSH. OSamp was added into the GSH solution (350 µM) with or without esterase. Figure 3B shows the level of GSH after 3 h of incubation. In the absence of esterase, the addition of OSamp caused no change in the GSH level. However, in the presence of esterase,


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OSamp depleted GSH in a concentration-dependent manner suggesting that esterase attacks the carboxylic ester of OSamp to produce QM, leading to GSH depletion. Acid-responsiveness of OSamp was studied using NMR spectroscopy. After incubation at acidic pH (5.5), OSamp generated cinnamaldehyde, evidenced by the distinct appearance of aldehyde protons at ∼9.6 ppm (Figure 3C). The extent of acid-triggered cleavage of acetal increased with time. However, no change was observed after its incubation in phosphate buffer (pH 7.4), demonstrating that OSamp undergoes acid-triggered hydrolysis to generate cinnamaldehyde. OSamp was stable even in the presence of serum proteins for 3 days (Figure S3). These observations demonstrate that OSamp is stable under physiological conditions and could be activated by acidic pH and esterase to generate cinnamaldehyde and QM, respectively.

Figure 3. Dual stimuli-responsiveness of OSamp. (A) Dual stimuli-triggered hydrolysis of OSamp to generate two complementary pharmacophores. (B) GSH depletion by OSamp in the absence or persence of esterase. Values are mean±s.d. (n=4). ***p

Dual Stimuli-Activatable Oxidative Stress Amplifying Agent as a Hybrid

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