Identification of Catechol-Type Diphenylbutadiene as a Tyrosinase

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Functional Structure/Activity Relationships

Identification of a catechol-type diphenylbutadiene as a tyrosinase-activated pro-oxidative chemosensitizer against melanoma A375 cells via GST inhibition Yuan Ji, Fang Dai, Shuai Yan, Jing-Yang Shi, and Bo Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02875 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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Journal of Agricultural and Food Chemistry

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Identification of a catechol-type diphenylbutadiene as a

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tyrosinase-activated pro-oxidative chemosensitizer against

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melanoma A375 cells via GST inhibition

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Yuan Ji, Fang Dai, Shuai Yan, Jing-Yang Shi, Bo Zhou*

6

State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222

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Tianshui Street S., Lanzhou, Gansu 730000, China

8 9 10 11 12 13 14 15

_________________

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*Corresponding author.

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State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222

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Tianshui Street S., Lanzhou, Gansu 730000, China

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E-mail: [email protected]

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Tel: +86-931-8912500

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Fax: +86-931-8915557

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ORCID: Bo Zhou: 0000-0002-8713-5906 1

ACS Paragon Plus Environment


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Abstract

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Glutathione S-transferases (GSTs) play an active role in the development of

25

drug-resistance by numerous cancer cells including melanoma cells, which is a major

26

cause of chemotherapy failure. As part of our continuous effort to explore why dietary

27

polyphenols bearing the catechol moiety (dietary catechols) show usually anticancer

28

activity, a catechol-type diphenylbutadiene (3,4-DHB) was selected as a model of

29

dietary catechols to probe whether they work as pro-oxidative chemosensitizers via

30

GST inhibition in melanoma cells. It was found that in human melanoma A375 cells,

31

3,4-DHB is easily converted into its ortho-quinone via copper-containing

32

tyrosinase-mediated two-electron oxidation along with generation of reactive oxygen

33

species (ROS) derived from the oxidation; the resulted ortho-quinone and ROS are

34

responsible for its ability to sensitize the cisplatin-resistant cells by inhibiting GST

35

followed by induction of apoptosis in a ASK1-JNK/p38 signaling cascade and

36

mitochondria-dependent pathway. This work provides further evidence to support that

37

dietary

38

tyrosinase-dependent pro-oxidative role, and gives useful information for designing

39

polyphenol-inspired GST inhibitors and sensitizers in chemotherapy against

40

melanoma.

catechols

exhibit

anti-melanoma

activity

by

virtue

of

their

41 42

Keywords: glutathione S-transferase, catechols, tyrosinase, cisplatin resistance,

43

sensitizer

44 2


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45

Introduction

46

Glutathione-S-transferases (GSTs, EC 2.5.1.18) are a family of phase II

47

detoxification enzymes that catalyze the conjugation of reduced glutathione (GSH)

48

with electrophilic xenobiotic substrates, making them more water-soluble to promote

49

their efflux from cells.1,2 The GST family in mammals is comprised of at least seven

50

distinct classes of cytosolic GSTs, namely, alpha (A), mu(M), pi (P), sigma (S), zeta

51

(Z), theta (T) and omega (O), but they share the structural similarity in the active sites:

52

a specific GSH binding site (G-site) and a hydrophobic site (H-site) which binds a

53

wide variety of electrophiles.1,3 Overexpression of GSTs has been identified in a

54

plethora of cancer cells and contributes significantly to their development of

55

resistance towards anticancer drugs (such as cisplatin and doxorubicin).4-9 Therefore,

56

GSTs are regarded as attractive targets for developing their inhibitors as cancer

57

chemosensitizers.10-14

58

Melanoma is a highly metastatic and lethal cancer of skin15,16 and is characterized

59

of overexpression of tyrosinase (TYR), a copper-containing enzyme which is

60

responsible for catalyzing the production of melanin from tyrosine.17 High mortality

61

in melanoma patients is partly due to resistance to chemotherapy. Increased

62

expression and activity of GST-pi were also observed in melanoma cells,18-22 it is thus

63

of importance to develop novel GST inhibitors for sensitizing melanoma cells to

64

chemotherapy agents.

65

The catechol skeleton is widely found in dietary polyphenols, such as caffeic acid

66

phenethyl (CAPE), epigallocatechin gallate, quercetin, luteolin, piceatannol and so on. 3



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These dietary catechols show usually potent anticancer activity including inhibitory

68

activity against GSTs.23-25 It is known that catechols are very easily converted to

69

o-quinones together with generation of reactive oxygen species (ROS) via two-step

70

single-electron oxidation in the presence of metal ions such as Cu(II) ions.26 In

71

contrast, the mechanism by which the copper-containing TYR mediates the

72

conversion is different; generally speaking, this conversion does not produces any

73

ROS but water as the only product of oxygen reduction, in a cycle involving oxidation

74

of two molecules of catechol per oxygen molecule.17 However, it is also suggested

75

that ROS (including superoxide anion radicals, hydrogen peroxide, hydroxyl radicals

76

and single oxygen) could be generated from the TYR-mediated oxidation of tyrosine

77

and the catechol-type L-dopa,27-29 probably due to the electron leakage from the

78

four-electron reduction of oxygen to water. Additionally, in biological system,

79

o-quinones are redox-active to generate ROS through redox cycling of o-quinones and

80

semiquinone radical anions.30 Thus, among all the factors determining anticancer

81

activity of dietary catechols, their oxidative conversion (pro-oxidative role) might be

82

critical because multiple signaling pathways can be modulated by the resulting

83

o-quinones or ROS via covalent or oxidative modification of critical cysteine residues

84

on redox-sensitive target proteins.24-26,30-34 For example, luteolin24 and CAPE25 have

85

been identified as potent GST inhibitors in melanoma cells depending on their

86

oxidative conversion into o-quinones mediated by TYR.

87

To understand why dietary catechols show usually potent anticancer activity, we

88

used previously a catechol-type stilbene (3,4-dihydroxy-trans-stilbene, 3,4-DHS, 4


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Scheme 1) as a model molecule to investigate the related mechanisms.26,31-33 3,4-DHS

90

is an analog of resveratrol (RES), a natural antioxidant derived from grapes, but also

91

bears the same catechol moiety as that of natural piceatannol. It was found that

92

depending on its catechol moiety, 3,4-DHS is capable of exploiting intracellular

93

copper ions to in situ generate o-quinone and ROS, resulting in activation of nuclear

94

factor erythroid-2 related factor 2 (Nrf2)31 and inhibition of angiogenesis.32

95

Furthermore, we designed recently a catechol-type diphenylbutadiene (3,4-DHB,

96

Scheme 1) by elongating the conjugated link of 3,4-DHS to facilitate the generation of

97

o-quinone, and found that 3,4-DHB is a more potent anti-melanoma agent than

98

3,4-DHS by constructing an efficient catalytic redox cycle with TYR and NAD(P)H:

99

quinone oxidoreductase-1 to induce H2O2-driven death of melanoma B16F1 cells.33

100

The latter case suggest that dietary catechols might be TYR-dependent pro-oxidative

101

anticancer agents in melanoma cells. To further verify this hypothesis, herein we used

102

the above molecules (RES, 3,4-DHS and 3,4-DHB) and cisplatin-resistant human

103

melanoma A375 cells to probe whether dietary catechols work as TYR-dependent

104

pro-oxidative chemosensitizer via GST inhibition, and the detailed mechanisms.

105

Among the molecules tested, RES was employed as a negative control to emphasize

106

the importance of the catechol moiety in melanoma chemosensitization, and 3,4-DHB

107

was applied to examine how elongation of the conjugated link affects the

108

chemosensitization.

109

Scheme 1 here

110 5



111

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Materials and methods

112

Materials. The RES and its catechol-type analogs (3,4-DHS and 3,4-DHB) were

113

synthesized according to our previous papers.35-37 Dithiothreitol (DTT), TYR from

114

mushroom (7164 U/mg), Glutathione S-Transferase from equine liver, cisplatin,

115

methyl

116

2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from

117

Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM)

118

was obtained from Gibco (USA). FITC Annexin V apoptosis Detection Kit was

119

purchased from BD Biosciences (USA). The primary antibody against β-actin was

120

obtained from Santa Cruz Biotechnology (USA) and the other primary antibodies

121

against caspase 3, caspase 9, poly (ADP-ribose) polymerase-1 (PARP-1), cytochrome

122

c, Bcl-2, Bax, apoptosis signal-regulating kinase 1 (ASK1), c-jun N-teiminal kinase

123

(JNK), p-JNK, p38 and p-p38 were obtained from Cell Signaling Technology

124

(Beverly, MA, USA). Horseradish peroxidase (HRP)-labeled secondary antibody was

125

obtained from TransGen Biotech (Beijing, China). IP lysis buffer and BCA Protein

126

Assay Kit were purchased from Beyotime (Jiangsu, China).

thiazolyl

tetrazolium

(MTT),

copper

chloride

and

127

Assay for in vitro GST-inhibitory activity. The GST-inhibitory activity of RES

128

and its catechol-type analogs was measured according to the method reported by Ang

129

et al38 with a few modifications. Briefly, equine liver GST (2.5 µg/mL) was incubated

130

with the tested compounds (6.25, 12.5, 25, 50 µM) with or without cupric chloride (50

131

μM) or TYR (2.5 U/mL, 0.003 µM) in the absence and presence of DTT (400 μM) or

132

caoptopril (100 μM) in 100 mM phosphate buffer (pH 6.5) in containing 1 mM GSH 6


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133

(final

volume

300

µL)

at

37

134

1-chloro-2,4-dinitroenzene (CDNB) with the final concentration of 1 mM, the

135

absorbance of the tested solution was measured at 340 nm for 180 s (interval = 30 s)

136

on a multimode reader (Infinite M200, TECAN) at 25 ℃.

℃

for

30

min.

After

addition

of

137

UV/Vis spectral measurements. UV/Vis spectra of 3,4-DHB (30 µM) in the

138

absence and presence of cupric chloride (30 μM) or TYR (8300 U/mL, 9.73 µM) in

139

CH3CN/water (1/1, v/v) at ambient temperature were acquired by a TU-1901

140

spectrophotometer (Persee, Beijing, China, 1 cm quartz cell). When necessary, DTT

141

(400 µM) was introduced into the reaction system. All the spectra were run against

142

blanks containing CH3CN/water (1/1, v/v).

143

Cell culture. Human melanoma A375 cells were obtained from National

144

Infrastructure of Cell Line Resource (Beijing, China). The cells were cultured in

145

DMEM medium with 10% fetal bovine serum (purchased from Royacel, Lanzhou,

146

China), penicillin (100 unites/mL) and streptomycin (100 unites/mL), and maintained

147

at 37 ℃ in an incubator with a humidified atmosphere containing 5% CO2.

148

Cell viability assay. A375 cells (4 × 103 cells/well) were seeded in 96-well plates

149

with DMEM medium for 24 h followed by replacement with fresh medium and

150

treatment with the tested compounds for 48 h with and without pretreatment of DTT

151

(400 µM) or captopril (100 µM) for 1 h . Subsequently, the medium was replaced

152

with fresh medium containing MTT (0.5 mg/mL). After incubation for 4 h, DMSO

153

(100 L) was added to each well. The values of optical density at 570 nm were

154

measured on a microplate reader (Bio-Rad M680, USA). 7



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155

Imaging of intracellular GST activity. A fluorogenic substrate for imaging

156

intracellular GST activity, 2,4-dinitrobenzenesulfonamide cresyl violet (DNs-CV),

157

was synthesized according to the previous reference.39 A375 cells (1 × 105 cells/mL)

158

were seeded in 6-well plates and cultured for 24 h. Then the cells were treated with

159

RES and its catechol-type analogs (8 µM) for 5 h with or without pretreatment with

160

DTT (400 µM) or captopril (100 µM) for 1 h followed by replacement with the fresh

161

medium containing DNs-CV (2 M) and incubation for another 30 min in dark at

162

37 ℃. The cells were washed three times with PBS and imaged by fluorescence

163

microscope (Leica DM 4000B, Leica Microsystems Inc. United States) with a × 20

164

objective lens.

165

Imaging of ROS accumulation. The ROS accumulation in A375 cells was imaged

166

by the fluorescent probe DCFH-DA.40 A375 cells (2×105 cells/well) were pretreated

167

with DTT (400 μM) or captopril (100 μM) for 1 h, treated with 3,4-DHB (8 μM) for

168

12 h, and then washed three times with PBS followed by incubation with DCFH-DA

169

(5 μM) for 30 min at 37 ℃. Subsequently, the cells were washed again with PBS for

170

three times. The fluorescence images were acquired by microscope (Leica DM

171

4000B, Germany) with a ×20 objective lens.

172

Apoptosis analysis. A375 (1 × 105 cells/mL) were seeded in six-well plates and

173

treated with 3,4-DHB, or/and cisplatin for 46 h in the absence and presence of

174

pretreatment with DTT (400 µM) or captopril (100 µM) for 1 h. Then the cells were

175

harvested and analyzed by a FACS Canto flow cytometer (BD Biosciences, USA).

176 8


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177

Western blotting analysis. A375 (2 × 106 cells/dish) were treated with 3,4-DHB

178

or/and cisplatin for 48 h with or without pretreatment with DTT (400 µM) or captopril

179

(100 µM) for 1 h. Subsequently, the cells were washed twice with ice-cold PBS and

180

lysed with ice-cold IP lysis buffer for 5 min. The lysates were collected and

181

centrifuged at 14,000 g for 10 min at 4 °C. The supernatant (protein) was collected

182

and the protein concentrations were quantified by the BCA Protein Assay Kit. Equal

183

amounts (40 μg) of protein sample were resolved by 15 % SDS-PAGE gels,

184

transferred to nitrocellulose membranes (0.45 µm, Bio-Rad Laboratories, Hercules,

185

CA, USA), and blocked with 5 % non-fat milk. After that, the membranes were

186

incubated with specific antibodies against caspase 3, caspase 9, Bcl-2, Bax,

187

cytochrome c, PARP-1, ASK1, JNK, p-JNK, p38 and p-p38 at 4 °C overnight. The

188

nitrocellulose membrane was washed three times with TBST buffer (50 mM

189

Tris-HCl, 150 mM NaCl and 0.1 % Tween-20) and incubated with HRP-conjugated

190

secondary antibody for 1 h at room temperature. The membrane was also washed

191

three times as described above and then covered with the solution of an ECL Western

192

blotting detection kit (Amersham Co., Bucks, U.K.). The chemiluminescent detection

193

was carried out on a chemiluminescence system (ImageQuant 400, USA).

194

Statistical analysis. Data were expressed as mean ± SD from at least three

195

independent experiments. Significant differences (P < 0.05) between the means of two

196

groups were analyzed by Student’s t-test of the SPSS software (version 22).

197 198 9



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199

Results and discussion

200

Structure-activity relationship (SAR) of RES and its catechol-type analogs as in

201

vitro GST inhibitors. We first evaluated the SAR of RES and its catechol-type

202

analogs as GST inhibitors by monitoring their inhibitory effect on conjugation of

203

CDNB to GSH catalyzed by equine liver GST,41 which is commercially available

204

and low-cost, also share a homologous structure of human GST-alpha.42 To clarify the

205

contribution of their oxidative conversion by TYR to their GST-inhibitory activity,

206

TYR was introduced into the tested solution. Considering that TYR is a

207

copper-containing enzyme, we also used cupric chloride to compare its efficacy with

208

that of TYR in oxidizing RES and its catechol-types analogs. As shown in Figure 1A

209

and B, neither RES alone nor RES in the presence of cupric chloride or TYR showed

210

obvious effect on the GST inhibition. In contrast, its catechol-type analogs (3,4-DHS

211

and 3,4-DHB) alone inhibited dose-dependently the GST activity due to their

212

auto-oxidation, and the presence of cupric ions or TYR intensified remarkably the

213

inhibitory effect (Figure 1A and B). These results indicate that although RES can be

214

oxidized by cupric ions43 or TYR,44 its conversion rate into o-quinone is far below

215

that achieved in the case of 3,4-DHS and 3,4-DHB. A comparison of Figure 1A with

216

1B also highlight that TYR (0.003 μM) mediates more effectively the oxidation of

217

3,4-DHS and 3,4-DHB than cupric ions (50 µM), leading to more active inhibition

218

against the GST. Additionally, 3,4-DHB is a more effective GST inhibitor than

219

3,4-DHS in the absence and presence of cupric ions or TYR. This can be easily

220

rationalized by the fact that elongation of the conjugated link decreases effectively the 10


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221

oxidation potential of 3,4-DHS to facilitate the generation of o-quinone and ROS. To

222

further clarify this point, DTT (an effective quencher of either electrophilic o-quinone

223

or ROS) and captopril (an inhibitor of TYR) were applied. Addition of DTT reversed

224

significantly the ability of 3,4-DHB plus cupric ions or TYR to inactivate GST(Figure

225

1C and D), and introduction of captopril also revised its ability to inactivate GST in

226

the presence of TYR (Figure 1C), highlighting again that its oxidative conversion into

227

o-quinone is critical for its ability to inactivate GST. It should be pointed out that

228

either DTT or captopril could not completely reverse the ability of 3,4-DHB plus

229

cupric ions or TYR to inactivate GST, probably due to its auto-oxidative ability and

230

the fact that its adduct with DTT or GSH in the tested solution still possesses some

231

extent of inhibitory activity against the GST.

232

Figure 1 here

233

Evidence for oxidative conversion of 3,4-DHB into o-quinone. Most o-quinones

234

are short-lived and cannot be effectively separated. To provide evidence to support

235

oxidative conversion of 3,4-DHB into o-quinone mediated by cupric ions or TYR, we

236

examined subsequently its cupric ion- or TYR-induced UV-visible absorption changes

237

in CH3CN/water (1/1, v/v) under aerobic conditions. It can be seen from Figure 2A

238

and B that addition of either cupric ions or TYR induced a prompt decrease in the

239

maximum absorbance of 3,4-DHB centered at 342 nm accompanied by the

240

development of a new band at about 504 nm. The new band corresponds to the

241

formation of o-quinone of 3,4-DHB, because it can be smoothened by introducing

242

nucleophilic DTT. This is the reason why DTT is capable of revising the ability of 11



243

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3,4-DHB plus cupric ions or TYR to inactivate GST. Figure 2 here

244 245

SAR of RES and its catechol-type analogs as chemosensitizers against A375

246

cells via GST-inhibition. It is well-known that overexpression of GSTs is closely

247

related to resistance of cancer cells to cisplatin because they catalyze the conjugation

248

of cisplatin with GSH to promote efflux of cisplatin from cancer cells and to thereby

249

intercept formation of cisplatin-DNA adducts.45 With the in vitro GST-inhibitory data

250

in hand, we wondered whether RES and its catechol-type analogs can sensitize human

251

melanoma A375 cells resistant to cisplatin. When A375 cells were treated with serial

252

concentrations of cisplatin (1, 2, 4, 6, 8 and 10 µM) in the absence and presence of

253

RES and its catechol-type analogs, 3,4-DHB exhibited the strongest synergistic effect

254

with cisplatin in killing A375 cells, followed by 3,4-DHS, while RES failed to do this

255

(Figure 3A-C). For example, 3,4-DHB (8 µM) alone was almost nontoxic (the cell

256

viability being 89.2 ± 2.7 %), and cisplatin (8 µM) alone induced death of almost half

257

of cells (the cell viability being 53.0 ± 1.3 %), but combination of the two triggered

258

death of most cells (the cell viability being 24.7 ± 1.8 %). Furthermore, the synergistic

259

killing of A375 cells by 3,4-DHB plus cisplatin was abrogated by pretreatment of the

260

cells with either DTT or captopril (Figure 4A and B), suggesting that its intracellular

261

oxidative conversion into o-quinones mediated by TYR along with generation of ROS

262

from the oxidation is responsible for its ability to sensitize cisplatin-resistant A375

263

cells.

264

Figures 3 and 4 here 12


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Noticeably, the activity order of RES and its catechol-type analogs as

266

chemosensitizers coincides with their ability to inactivate equine liver GST, hinting

267

the possibility that they work as chemosensitizers via intracellular GST inhibition. To

268

clarify this point, we employed a fluorogenic substrate (DNs-CV) developed by

269

Zhang et al 39 to image the GST activity in living A375 cells. DNs-CV is essentially

270

non-fluorescent, but emits red fluorescence upon the GST-catalyzed cleavage of the

271

dinitrobenzenesulfonamide by GSH.39 As illustrated in Figure 5A, red fluorescence

272

signal was observed when the cells were incubated with DNs-CV (2 µM) for 30 min.

273

However, this signal was significantly inhibited by pretreating with 3,4-DHB (8 µM)

274

for 5 h (Figure 5D). By comparison, under the same conditions, 3,4-DHS inhibited

275

marginally this signal (Figure 5C), and RES was inactive in inhibiting this signal

276

(Figure 5B). More importantly, the fluorescence intensity weakened by 3,4-DHB

277

(Figure 5D) returned back to its original level based on the pretreatment with either

278

DTT (Figure 5F) or captopril (Figure 5H). These results further support that after its

279

bioactivation mediated by TYR, 3,4-DHB inhibits intracellular GSTs to sensitize

280

cisplatin-resistant A375 cells.

281

Figure 5 here

282

TYR-dependent ROS accumulation induced by 3,4-DHB in A375 cells.

283

3,4-DHB is itself not an electrophile, but its oxidative product, o-quinones, are soft

284

electrophiles capable of covalently modifying critical cysteine residues (soft

285

nucleophiles) on intracellular GSTs, resulting in their inactivation. On the other hand,

286

in the reductive cytoplasm, o-quinones are readily converted into semiquinone radical 13



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287

anions via single-electron reduction;30 These intermediates is highly active and can be

288

oxidized back to o-quinones by oxygen, leading to generation of the superoxide anion

289

radicals.30 Moreover, various ROS are suggested to be generated from the

290

TYR-mediated oxidation of tyrosine and the catechol-type L-dopa.27-29 We have

291

recently found that 3,4-DHB can promote TYR-dependent H2O2 accumulation to kill

292

melanoma B16F1 cells selectively.33 Considering that ROS could be also factors to

293

inactivate intracellular GSTs via oxidative modification of their critical cysteine

294

residues, we used the DCFH-DA probe to image the ROS accumulation in A375 cells

295

induced by 3,4-DHB.40 When the cells were treated with 3,4-DHB for 12 h followed

296

by incubation with DCFH-DA for 0.5 h, the green fluorescence appeared (Figure 6),

297

suggesting generation of ROS. Parallel to their reversion on the intracellular

298

GST-inhibitory activity of 3,4-DHB (Figure 5), and the synergistic cytotoxicity of

299

3,4-DHB plus cisplatin (Figure 4), addition of DTT and captopril weakened

300

significantly the increased fluorescence signal (Figure 6). Taken together, these

301

results support that intracellular generation of ROS depends on the TYR-mediated

302

oxidization of 3,4-DHB, and contributes to its chemosensitization against A375 cells

303

via GST inhibition.

304

Figure 6 here

305

Apoptosis of A375 cells synergistically induced by 3,4-DHB plus cisplatin via

306

activation of ASK1-JNK/p38 signaling. To rationalize the detailed mechanisms by

307

which 3,4-DHB plus cisplatin kill synergistically A375 cells, we also performed

308

apoptosis analysis by using flow cytometry. Consistent with the results of 14


Page 15 of 33


309

cytotoxicity, 3,4-DHB plus cisplatin triggered synergistically apoptosis of A375 cells

310

(Figure 7). Specifically, the percentages of cells in early and late apoptosis for

311

3,4-DHB (8 µM), cisplatin (8 µM) and combination of the two were 0.4% and 7.1%,

312

6.5% and 37.5%, and 4.9% and 62.5%, respectively. The dependency of apoptosis on

313

mitochondria was subsequently verified by Western blotting. Figure 8 shows that in

314

A375 cells, 3,4-DHB plus cisplatin induced synergistically down-regulation of

315

anti-apoptotic Bcl-2, up-regulation of pro-apoptotic Bax, release of cytochrome c

316

from mitochondria into cytosol, activation of caspase-9, and -3 and cleavage of Poly

317

(ADP-ribose) polymerase-1 (PARP-1). As expected, pretreatment with either DTT or

318

captopril revised again the apoptosis and the change of the related protein expression

319

induced synergistically by 3,4-DHB plus cisplatin (Figures 7 and 8).

320

Figures 7 and 8 here

321

Besides their enzymatic role, various GSTs have been suggested to bind and inhibit

322

the activity of kinases such as JNK146,47 and its upstream ASK1,48 one of MAP3K

323

family members, leading to inhibition of apoptosis. This is another reason why

324

overexpression of GSTs is associated with resistance of cancer cells to apoptosis

325

induced by various anticancer drugs, even including those being not effective

326

substrates of GSTs. Thus, we finally asked whether inhibition of GST by 3,4-DHB

327

activates ASK1 signal. Western blotting experiments illustrate that 3,4-DHB plus

328

cisplatin increased synergistically the expression of ASK1, p-JNK and p-p38 in A375

329

cells, but the increased protein expression was abrogated by pretreatment the cells

330

with either DTT or captopril (Figure 9). Together, based on the above experiments 15



Page 16 of 33

331

(Figures 5-9) we can conclude that depending on its oxidative conversion into

332

o-quinones mediated by TYR together with generation of ROS, 3,4-DHB works as a

333

potent GST inhibitor to sensitize cisplatin-resistant A375 cells via induction of

334

apoptosis in a ASK1-JNK/p38 signaling cascade and mitochondria-dependent

335

pathway (Scheme 2). Figure 9 and Scheme 2 here

336 337

In summary, taking 3,4-DHB as a model of dietary catechols, we identified this

338

molecule as a tyrosinase-dependent pro-oxidative GST inhibitor to sensitize

339

cisplatin-resistant A375 cells. This work gives added confidence for dietary catechols

340

acting as pro-oxidative anti-melanoma agents and supports for the catechol moiety

341

being a key structural determinant to develop GST inhibitors and sensitizers in

342

chemotherapy against melanoma.

343 344

Funding

345

This work was supported by the National Natural Science Foundation of China (Grant

346

Nos. 21672091) and the 111 Project.

347 348

Notes

349

The authors declare no conflict of interest.

350 351 352 16


Page 17 of 33


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507 508

Figure legends

509

Scheme 1. Molecular structures of RES and its catechol-type analogs.

510 511

Scheme 2. Proposed mechanisms by which 3,4-DHB work as tyrosinase

512

(copper)-dependent pro-oxidative chemosensitizer in the cisplatin-resistant A375 cell.

513 514

Figure 1. The in vitro GST-inhibitory activity of RES and its catechol-type analogs

515

in the absence and presence of cupric chloride (50 µM) (A) or TYR (2.5 U/mL, 0.003

516

µM) (B), and revision of DTT and captopril on the activity of 3,4-DHB plus cupric

517

ions (C) or TYR (D). *p < 0.05, **p < 0.01, ***p < 0.001; #p < 0.05, ###p < 0.001.

518 519

Figure 2. Absorption spectral changes of 3,4-DHB (30 µM) in the absence and

520

presence of cupric chloride (30 µM) (A) or TYR (8300 U/mL, 9.73 μM) (B) in

521

CH3CN/water (1/1, v/v) at ambient temperature. The dotted lines show the quenching

522

of o-quinone by DTT (400 μM).

523 524

Figure 3. Effect of RES (A), 3,4-DHS (B) and 3,4-DHB (C) on the death of A375

525

cells induced by different concentrations of cisplatin. *p < 0.05, **p < 0.01, ***p

Identification of Catechol-Type Diphenylbutadiene as a Tyrosinase

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