Protective Effects of Minor Components of Curcuminoids on Hydrogen

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Protective Effects of Minor Components of Curcuminoids on Hydrogen Peroxide-Treated Human HaCaT Keratinocytes Yuh-Hwa Liu, Yin-Shiou Lin, Yu-Wei Huang, Sheng-Uei Fang, Shyr-Yi Lin, and Wen-Chi Hou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01196 • Publication Date (Web): 20 Apr 2016 Downloaded from http://pubs.acs.org on April 20, 2016

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Journal of Agricultural and Food Chemistry

Protective Effects of Minor Components of Curcuminoids on Hydrogen

1

Peroxide-Treated Human HaCaT Keratinocytes

2 3

¶,▓

4

Yuh-Hwa Liu,

5 6 7 8

Shyr-Yi Lin, ¶

▓,*

and Wen-Chi Hou†,*

Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan

▓

9

Department of General Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan

10

†

11

▼

12

⊗

13

Yin-Shiou Lin,† Yu-Wei Huang,† Sheng-Uei Fang,▼,⊗

Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan Division of Gastroenterology, Taipei Medical University Hospital, Taipei 110, Taiwan

Department of Internal Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan

14 15 16 17

*To whom correspondence should be addressed:

18

Prof. Hou, Wen-Chi

19

Fax: 886 (2) 2378-0134; E-mail: [email protected]

20

Or

21

Prof. Lin, Shyr-Yi

22

E-mail: [email protected]

23 24

1

ACS Paragon Plus Environment


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25

ABSTRACT

26

Hydrogen peroxide, one of the reactive oxygen species (ROS), can cause intracellular oxidative

27

stress associated with skin aging and/or photo-aging. Curcumin, a polyphenol in turmeric, has been

28

reported to exhibit biological activity. In this study, five naturally occurring curcuminoids

29

[curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), monohydroxy-DMC

30

and

31

hydrogen-peroxide-induced oxidative stress in the immortalized human keratinocyte cell lines

32

(HaCaT cells). These five curcuminoids at 10 µM, but not at 5 µM, were showed to exhibit

33

cytotoxicities toward HaCaT keratinocytes. Therefore, 5 µM of five curcuminoids were selected for

34

further investigations. Cells were pretreated with or without curcuminoids for 2.5 h before 24-h

35

hydrogen

36

monohydroxy-DMC or monohydroxy-BDMC, but not curcumin, DMC and BDMC, showed

37

protective activity elevating cell viability compared to those with direct hydrogen peroxide

38

treatments. Pretreatments of monohydroxy-DMC and monohydroxy-BDMC showed the best

39

protective effects to reduce apoptotic cell populations and intracellular ROS by flow cytometry as

40

well as the changes of mitochondrial membrane potential compared to those with direct hydrogen

41

peroxide treatments. The pretreatments of monohydroxy-DMC and monohydroxy-BDMC reduced

42

c-jun and c-fos mRNA expressions and p53 tumor suppressor protein expressions, and increased

43

HO-1 protein expressions and glutathione peroxidase (GPx) activities, respectively, compared to

44

those with direct hydrogen peroxide treatments. The five curcuminoids exhibited similar hydrogen

45

peroxide scavenging activity in vitro. It was proposed that monohydroxy-DMC and

46

monohydroxy-BDMC could induce higher levels of antioxidant defense systems than curcumin,

47

DMC or BDMC could against hydrogen peroxide-induced oxidative stress and apoptosis of HaCaT

monohydroxy-BDMC]

peroxide

(150

were

µM)

used

to

treatments.

investigate

their

Pretreatments

of

protective

minor

roles

against

components

of

2


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48

keratinocytes, and that they may have potential as ingredients in the development of anti-aging

49

cosmetics for skin care.

50

Keywords: curcuminoids, hydrogen peroxide, HaCaT keratinocyte, oxidative stress, reactive

51

oxygen species (ROS)

52 53

█ INTRODUCTION

54

Keratinocytes and melanocytes are the two main cell populations in the epidermis, the outer layer

55

of the skin. Keratinocytes represent almost 95% of the cells present in the epidermal layer.1 Not

56

only is the skin the first natural barrier against physical, chemical and microbial attacks, but it also

57

defends against sunlight’s ultraviolet (UV) radiation.1 While UV radiation can induce DNA

58

damage in the epidermal cells;, the melanin pigments that are produced by melanocytes in the basal

59

layer of the epidermis act as natural protectors to prevent UV-induced nuclear DNA damage to the

60

skin cells.2–4 Each melanocyte attached to the epidermal basement membrane exports mature

61

melanosomes to nearby keratinocytes through its dendrites. The uptake of melanosomes by the

62

keratinocytes is an active process involving the dendrites and filopodia of the melanocyte, as well

63

as regulatory processes in the keratinocytes.2–4 It was proposed that sunlight radiation could induce

64

DNA mutation, photo-aging, lipid peroxidation, inflammatory cytokines and cell apoptosis in

65

human or animal skin directly by repeated exposures5, or indirectly through the generation of

66

intracellular reactive oxygen species (ROS) 5–7 such as superoxide radicals, hydroxyl radicals and

67

hydrogen peroxide.8 Therefore, keratinocytes in the epidermis are the main targets under sunlight,

68

and hydrogen peroxide is produced in the cells after sunlight UVB irradiation, which may elevate

69

cellular oxidative stress and enhance cell apoptosis and carcinogenesis.6 It was reported that oral

70

and topical application of green tea polyphenols before UVB radiation could protect against skin

71

cancer development in mice, and the mechanism might be linked to antioxidant activities of green 3



72

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tea polyphenols as radical scavengers.6, 9

73

Turmeric, derived from the rhizome of Curcuma longa, is a native plant in tropical South Asia,

74

and is widely used as a dietary spice and traditional medicine.10–12 Turmeric has traditionally been

75

used to treat a variety of diseases and conditions, including those of the skin, the pulmonary and

76

gastrointestinal systems, aches, pains, wounds, sprains and liver disorders ever since the

77

development of India’s Ayurveda (1900 BC)13, which posits the curcuminoids and sesquiterpenes

78

to

79

[1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, Figure 1], and its two major

80

structural

81

1-(4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione]

82

bisdemethoxycurcumin (3) [BDMC, 1,7-bis(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione], which

83

collectively are called curcuminoids.14 The curcumin commercial preparations generally contain

84

77% curcumin, 17%–18% DMC and 5%–6% BDMC.13, 15 Low amounts of curcumin analogs are

85

found

86

[1-(3,4-dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione],

87

monohydroxy-BDMC

(5)

88

[1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione],

and

89

tetrahydroxycurcumin17, which are also referred to as members of the curcuminoids. Curcumin has

90

been reported to have several biological activities14,15, such as antioxidant18–21, anti-inflammatory22,

91

neurite outgrowth promotion in vitro23, and improved effects against nonalcoholic fatty liver24,

92

diabetes or insulin resistance25–27, and obesity.28–29 Several reports have dealt with the use of

93

hydrogen peroxide to induce oxidative stress and/or cell death in HaCaT keratinocytes with or

94

without natural compound treatments: they were used to mimic the UV light’s direct and indirect

95

effects on keratinocytes in the skin’s epidermis.30–34 In this study, five naturally occurring

be

the

active

components.10–13

analogs,

in

Turmeric

contains

demethoxycurcumin

nature10,11,16,17,

such

as

curcumin

(2)

monohydroxy-DMC

(1)

[DMC, and

(4)

4


Page 5 of 37


96

curcuminoids (curcumin, DMC, BDMC, monohydroxy-DMC and monohydroxy-BDMC) were

97

used to investigate their protective roles against hydrogen peroxide-induced oxidative stress in the

98

immortalized human keratinocyte cell lines (HaCaT cells). Cells were pretreated with or without

99

curcuminoids for 2.5 h before 24-h hydrogen peroxide treatments to test their effects against

100

hydrogen-peroxide-induced oxidative stress and apoptosis of HaCaT keratinocytes. The five

101

curcuminoids exhibited similar hydrogen peroxide scavenging activity in vitro; however, the

102

monohydroxy-DMC and monohydroxy-BDMC showed better protective activities than did the

103

curcumin, DMC and BDMC in hydrogen peroxide-induced cell deaths in HaCaT cells. It was

104

proposed that monohydroxy-DMC and monohydroxy-BDMC could induce higher levels of

105

intracellular antioxidant defense systems than those of curcumin, DMC, or BDMC, and that they

106

may have potential as ingredients in the development of antioxidant, anti-radical, or anti-aging

107

cosmetics for skin care, which would require further investigation.

108 109 110

█ MATERIALS AND METHODS

111

Materials. The curcuminoids (Figure 1), including curcumin (1), demethoxycurcumin (2)

112

(DMC), bisdemethoxycurcumin (3) (BDMC), monohydroxy-DMC (4) and monohydroxy-BDMC

113

(5), were purchased from Laila Impex Co. Ltd. (Vijayawada, India) with purity higher than 99%.

114

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue, dimethyl

115

sulfoxide (DMSO), propidium iodide (PI), 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate

116

(DCFH-DA),

117

5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide (JC-1, T4069) were purchased

118

from Sigma Chemical Co. (St. Louis, MO). N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red,

119

InvitrogenTM) was purchased from Thermo Fisher Scientific Inc. (Rockford, IL). Dulbecco’s

hydrogen

peroxide

solution

(30%),

N-acetyl

cysteine

(NAC)

and

5



Page 6 of 37

120

modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL Co.

121

(NY, USA). The immortalized human keratinocyte cell lines (HaCaT cells) were kindly provided

122

by Prof. Sheen (National Taiwan University, Taipei, Taiwan). Heme oxygenase-1 (HO-1)

123

polyclonal antibody (rabbit, SPA-894: immunogen was recombinant human HO-1 protein) was

124

purchased from Assay Designs Co. (MI, USA), tumor suppressor protein p53 polyclonal antibody

125

(rabbit, No. 9282, immunogen was full-length human p53 fusion protein) was obtained from Cell

126

Signaling Technology, Inc. (MA, USA), and GAPDH monoclonal antibody (clone GAPDH-71.1)

127

was purchased from Sigma Chemical Co. (St. Louis, MO).

128 129

Cell Viability Determinations. The HaCaT keratinocytes were cultured in DMEM containing

130

10% FBS, and incubated at 37°C in a humidified atmosphere with 5% CO2. For a cell viability

131

assay, 100 µL of HaCaT keratinocytes (1×105/mL) were seeded onto a 96-well microtiter plate at

132

37°C in a humidified atmosphere with 5% CO2 for 24 h. The five curcuminoids (in DMSO), each at

133

a final concentration of 5 µM, were added and cultured at 37°C in a humidified atmosphere with

134

5% CO2 for another 24 h. For protection against hydrogen-peroxide-induced cell death, 100 µL of

135

HaCaT keratinocytes (1×105/mL) were seeded at 37°C in a humidified atmosphere with 5% CO2 for

136

24 h. The five curcuminoids (in DMSO), each at a final concentration of 5 µM, were added and

137

cultured at 37°C in a humidified atmosphere with 5% CO2 for 2.5 h. The medium was removed, and

138

then hydrogen peroxide at a final concentration of 150 µM was added and cultured for another 24 h.

139

The 0.4% DMSO was used as the control. After incubation, keratinocytes were incubated with

140

MTT (500 µg/mL) for 4 to 6 h to stain the cells.35 The absorbance at 570 nm was determined by

141

ELISA reader (TECAN Sunrise microplate reader, Männedorf, Switzerland). The results were

142

calculated and expressed as the cell viability by the following equation: (A570 of treated

143

sample)÷(A570 of the control) ×100%. 6


Page 7 of 37


144 145

Annexin V-FITC/PI Stains. The hydrogen-peroxide-induced cell deaths in keratinocytes with

146

or without curcuminoid pretreatments were stained with annexin V-FITC/PI and assayed by flow

147

cytometry.36 The HaCaT keratinocytes (1×105/mL) were seeded onto a 6-well plate at 37°C in a

148

humidified atmosphere with 5% CO2 for 24 h. The five curcuminoids (in DMSO), each at a final

149

concentration of 5 µM, were added and cultured at 37°C in a humidified atmosphere with 5% CO2

150

for 2.5 h. The medium was removed, and then hydrogen peroxide at a final concentration of 150

151

µM was added and cultured for another 24 h. Without hydrogen peroxide treatments, the 0.4%

152

DMSO was used instead of curcuminoids as the blank; with hydrogen peroxide treatments, the

153

0.4% DMSO was used instead of curcuminoids as the control. The treated HaCaT keratinocytes

154

were harvested, washed with PBS and then stained using FITC Annexin V Apoptosis Detection Kit

155

I (No. 556547, BD Biosciences, CA) following the manufacturer’s instructions, and analyzed by

156

flow cytometry (BD FACSCantoTM II, BD Biosciences, CA).

157 158

Intracellular ROS Measurements. The changes of intracellular peroxide levels in HaCaT

159

keratinocytes after hydrogen peroxide treatments with or without pretreatments of curcuminoids

160

were assessed by flow cytometry using DCFH-DA as a probe.37 The DCFH-DA penetrated into the

161

cells and was hydrolyzed by cellular esterase to DCFH, which was further oxidized by intracellular

162

peroxide into a strong fluorescent compound, dichlorofluorescein. The HaCaT keratinocytes

163

(1×105/mL) were seeded onto a 6-well plate at 37°C in a humidified atmosphere with 5% CO2 for

164

24 h. The five curcuminoids (in DMSO), each at a final concentration of 5 µM and

165

N-acetylcysteine (NAC, 10 mM, as the positive control), were added and cultured at 37°C in a

166

humidified atmosphere with 5% CO2 for 2.5 h. The medium was removed and washed. The cells

167

were then cultured for another hour after the addition of 10 µM DCFH-DA; hydrogen peroxide at a 7



Page 8 of 37

168

final concentration of 150 µM was then added and cultured for another hour. Without hydrogen

169

peroxide treatments, the 0.4% DMSO was used instead of curcuminoids as the blank; with

170

hydrogen peroxide treatments, the 0.4% DMSO was used instead of curcuminoids as the control.

171

The cells were washed twice with PBS and analyzed by flow cytometry (BD FACSCantoTM II, BD

172

Biosciences, CA) with excitation and emission settings of 488 nm and 530 nm, respectively. The

173

peroxide levels in the cells were plotted as one-parameter histograms with cell count on the y-axis

174

and fluorescence on the x-axis. The calculated mean value of the fluorescent distributions in the

175

fixed cell counts was expressed as the fluorescent intensity.

176 177

Mitochondrial Membrane Potentials Assays. The sensitive probe JC-138 was used to

178

evaluate the changes of mitochondrial membrane potential in HaCaT keratinocytes after hydrogen

179

peroxide treatments with or without pretreatments of curcuminoids. While JC-1 formed

180

J-aggregates in healthy cells of higher membrane potential with red fluorescence, JC-1 kept the

181

monomeric form in apoptotic cells of the lower membrane potential with green fluorescence38; the

182

ratio of green to red fluorescence revealed the status of cultured cells after treatments. The 100 µL

183

of HaCaT keratinocytes (1×105/mL) were seeded onto a 96-well plate at 37°C in a humidified

184

atmosphere with 5% CO2 for 24 h. The five curcuminoids (in DMSO), each at a final concentration

185

of 5 µM, were added and cultured at 37°C in a humidified atmosphere with 5% CO2 for 2.5 h, the

186

medium was removed, and then hydrogen peroxide at a final concentration of 150 µM, was added

187

and cultured for another 12 h. Without hydrogen peroxide treatments, the 0.4% DMSO was used

188

instead of curcuminoids as the blank; with hydrogen peroxide treatments, the 0.4% DMSO was

189

used instead of curcuminoids as the control. After incubation, the two-fold dilution of JC-1 (1

190

mg/mL) was added into a 96-well plate and cultured at 37°C in a humidified atmosphere with 5%

191

CO2 for 0.5 h. The red fluorescence (λex 550 nm and λem 600 nm) and green fluorescence (λex 485

192

nm and λem 535 nm) were detected by the VICTORTM X3 Multilabel Plate Reader (Perkin Elmer 8


Page 9 of 37


193

Inc.,

MA,

USA)

and

calculated

as

194

fluorescence/(RFUsample-RFUbackground) of red fluorescence.

(RFUsample-RFUbackground)

of

green

195 196

Glutathione Peroxidase Activity Determination. The changes of glutathione peroxidase

197

(GPx) activities in HaCaT keratinocytes after hydrogen peroxide treatments with or without

198

pretreatments of curcuminoids were determined by the ELISA assay kit. The HaCaT keratinocytes

199

(1×105/mL) were seeded onto a 6-well plate at 37°C in a humidified atmosphere with 5% CO2 for

200

24 h. Then, the monohydroxy-DMC (4) and monohydroxy-BDMC (5), each at a final concentration

201

of 5 µM, were added and cultured at 37°C in a humidified atmosphere with 5% CO2 for 24 h and

202

then

203

monohydroxy-BDMC (5), each at a final concentration of 5 µM, were pretreated for 2.5 h, the

204

medium was removed and washed, and hydrogen peroxide at a final concentration of 150 µM, was

205

then added and cultured for another 2 h. Without hydrogen peroxide treatment, the 0.4% DMSO

206

was used instead of curcuminoids as the blank; with hydrogen peroxide treatment, the 0.4% DMSO

207

was used instead of curcuminoids as the control. For GPx determination, cells were washed twice

208

with PBS, lysed and then determined by the Glutathione Peroxidase Assay Kit (No. 703102,

209

Cayman Chem. Co., Michigan, USA) using cellular GPx activity assays. The assay was based on a

210

coupled reaction with glutathione reductase to reduce the oxidized glutathione (GSSG) in the

211

presence of NADPH, with which the generated NADP+ showed a decrease in absorbance at 340

212

nm.

for

GPx

activity

determinations.

Alternatively,

monohydroxy-DMC

(4)

and

213 214

Effects of Curcuminoids on Hydrogen Peroxide Scavenging Activity in Vitro. The in vitro

215

hydrogen peroxide scavenging activities of each curcuminoid were determined by the amplex

216

red/horseradish peroxidase (HRP) system.39 The reaction mixture contained a final concentration of 9



Page 10 of 37

217

75 µM amplex red, 1 unit/mL HRP and 10 µM hydrogen peroxide in phosphate-buffered saline

218

without (as the control) or with 5 µM of each curcuminoid addition. After a 30-min reaction time,

219

the fluorescence (λex 530 nm and λem 590 nm) was detected by the VICTORTM X3 Multilabel Plate

220

Reader (Perkin Elmer Inc., MA, USA) and expressed as a fluorescent intensity (RFU).

221 222

RNA Extraction and qPCR for Gene Expressions. The HaCaT keratinocytes (1×105/mL)

223

were seeded onto a 6-well plate at 37°C in a humidified atmosphere with 5% CO2 for 24 h. The five

224

curcuminoids (in DMSO), each at a final concentration of 5 µM, were added and cultured at 37°C

225

in a humidified atmosphere with 5% CO2 for 2.5 h, the medium was removed, and hydrogen

226

peroxide at a final concentration of 150 µM was then added and cultured for another 24 h. Without

227

hydrogen peroxide treatments, the 0.4% DMSO was used instead of curcuminoids as the blank;

228

with hydrogen peroxide treatments, the 0.4% DMSO was used instead of curcuminoids as the

229

control. The treated HaCaT keratinocytes were harvested and washed with PBS. TRI reagent

230

(T9424, Sigma Chemical Co.), chloroform, and isopropanol were used to isolate RNA following the

231

manufacturer’s instructions. The purified RNA and oligo(dT), respectively, were used as the

232

template and primers to produce cDNA by SuperScript™ II reverse transcriptase and Platinum® Taq

233

DNA polymerase (Thermo Fisher Scientific Inc.). For quantification of specific gene expressions,

234

the real-time PCR in the Roche LightCycle 480 Real-Time PCR System (Roche Applied Science,

235

CA) was performed, and the cDNA, primers of the specific genes, and Power SYBR Green PCR

236

Master Mix (Thermo Fisher Scientific Inc.) were added for 40 cycles following the manufacturer’s

237

instructions. Each cycle consisted of 30 sec of denaturizing at 95°C, 60 sec of annealing at 58°C

238

and 60 sec of extension at 72°C. The primers of the specific genes were synthesized by Mission

239

Biotech.

240

GGATCAAGGCGGAGAGGAAG, reversed (20 mer) GCGTTAGCATGAGTTGGCAC; c-fos

Co.

(Taipei,

Taiwan)

as

follows40:

c-jun

(221

bp),

forward

(20

mer)

10


Page 11 of 37


241

(322

bp),

forward

(20

mer)

GGAGAATCCGAAGGGAAAGG,

242

GCTTGGGCTCAGGGTCATTG;

243

CGCTCTCTGCTCCTCCTGTT, reversed (20 mer) CCATGGTGTCTGAGCGATGT. These two

244

specific gene mRNA levels were normalized using the GAPDH mRNA level as an internal control.

245

The Ct (threshold cycle) was calculated in the intersection between an amplification curve and a

246

threshold line. The normalized specific gene expression level was calculated and expressed as

247

follows: ∆Ct1 = Ct(target genetreated) – Ct(GAPDHtreated); ∆Ct2 = Ct(target genecontrol) – Ct

248

(GAPDHcontrol); ∆∆Ct = ∆Ct1treated – ∆Ct2control, and the fold change of the specific gene was 2-∆∆Ct.

GAPDH

(80

bp),

reversed

forward

(20 (20

mer) mer)

249 250

Western Blot Analysis. Immune staining was performed to investigate the specific protein

251

expressions. The HaCaT keratinocytes (1×105/mL) were seeded onto a 6-well plate at 37°C in a

252

humidified atmosphere with 5% CO2 for 24 h. The five curcuminoids (in DMSO), each at a final

253

concentration of 5 µM, were added and cultured at 37°C in a humidified atmosphere with 5% CO2

254

for 2.5 h. The medium was removed, and then hydrogen peroxide at a final concentration of 150

255

µM was added and cultured for another 6 h or 24 h, respectively, for HO-1 or p53 expressions.

256

Without hydrogen peroxide treatments, the 0.4% DMSO was used instead of curcuminoids as the

257

blank; with hydrogen peroxide treatments, the 0.4% DMSO was used instead of curcuminoids as

258

the control. The treated HaCaT keratinocytes were harvested and then lyzed by cold RIPA buffer

259

containing a protease inhibitor cocktail (P-8340, Sigma Chemical Co.). Furthermore, the protein

260

contents in cell homogenates were quantified using the BCA Protein Assay Kit (Pierce

261

Biotechnology, Inc., Rockford, IL, USA). Equal protein amounts of cell homogenates were

262

subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After

263

electrophoresis, the gels were equilibrated with Tris-glycine buffer (pH 8.3) and transferred onto

264

immobile polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). The PVDF 11



Page 12 of 37

265

membranes were blocked with 1% gelatin in NaCl/EDTA/Tris (NET) solution for 1 h at room

266

temperature and incubated overnight at 4°C with each of the primary antibodies. The HO-1 and

267

tumor suppressor protein p53 antibodies were each used in a 1000-fold dilution; GAPDH antibody

268

was used in a 10000-fold dilution (in 0.25% gelatin in NET solution). The PVDF membranes were

269

washed thrice with phosphate-buffered saline Tween-20 (PBST) for 10 min. Thereafter, horseradish

270

peroxidase–conjugated IgG (goat anti-mouse or goat anti-rabbit IgG) solution (1000-fold dilution

271

in 0.25% gelatin in NET solution) was added, and the membrane was washed again using 1× PBST.

272

Immunoblots were detected using Western Chemiluminescent HRP Substrate kits containing

273

luminol reagents and peroxide solutions (No. WBKL S0050; Immobilon™, Millipore). Each blot

274

was imaged, and quantified and expressed as the relative density (%) by using the Syngene

275

G:bBOX imaging system (Syngene, UK) equipped with the GeneSnap software (Syngene, UK).

276

The relative density in the blank was considered as 100%.

277 278

Statistical Analyses. Data were expressed as mean ± SD. Multiple group comparisons were

279

performed using one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test;

280

these had not been indicated with the same alphabet and differed significantly (P < 0.05). The

281

differences of cell viability or GPx activity changes between curcuminoid treatments and the

282

control group were analyzed using Student’s t-tes; any difference in comparison with the control

283

group was considered statistically significant when P < 0.05 (*), or P < 0.01 (**), or P < 0.001

284

(***). Statistical analysis was performed using the GraphPad Prism 5.0 software (San Diego, CA,

285

USA).

286 287

█ RESULTS

12


Page 13 of 37


288

Effects of Curcuminoid Pretreatments on Hydrogen Peroxide-Induced Cell Death of

289

HaCaT Keratinocytes. Figure 1 shows the structures of five curcuminoids used in this study,

290

including curcumin (1), DMC (2), BDMC (3), monohydroxy-DMC (4), and monohydroxy-BDMC

291

(5). It was found that curcuminoids, each at a concentration of 5 µM (Figure 2A), showed no

292

significant toxicity (P > 0.05) compared to the control (DMSO, 0.4%) in cell viabilities of HaCaT

293

keratinocytes. While, the concentration was up to 10 µM (Figure 2B), each curcuminoids showed

294

significant toxicity (P < 0.05) compared to the control (DMSO, 0.4%) in cell viabilities of HaCaT

295

keratinocytes. Therefore, 5 µM of five curcuminoids were selected for further investigations. The

296

effects of curcuminoid pretreatments on hydrogen peroxide-induced cell death of HaCaT

297

keratinocytes were therefore investigated; the results are shown in Figure 2C. After having been

298

treated with 150 µM hydrogen peroxide for 24 h, the viabilities of HaCaT keratinocytes decreased

299

from 100% to 72.9 ± 2.1% and showed significant differences (P < 0.05) compared to the blank

300

(untreated cells). After having been pretreated with each curcuminoid for 2.5 h, the medium was

301

removed; the HaCaT keratinocytes were then treated with 150 µM hydrogen peroxide for 24 h. The

302

viabilities of HaCaT keratinocytes were 71.2 ± 4.0%, 58.0 ± 9.1%, 64.5 ± 4.2%, 83.8 ± 4.5% and

303

87.2 ± 6.4%, respectively. It was found that pretreatment of monohydroxy-DMC (4) and

304

monohydroxy-BDMC (5), but not curcumin (1), DMC (2), and BDMC (3), showed protective

305

effects against hydrogen peroxide toxicities to elevate the viabilities of HaCaT keratinocytes, and

306

showed significant differences (P < 0.05) compared to the control with direct hydrogen peroxide

307

treatments (Figure 2C).

308 309

Apoptosis. The hydrogen-peroxide-induced HaCaT keratinocyte cell death was determined by

310

flow cytometry with annexin V-FITC/PI double stains; in this process, cells were gated into four

311

quadrants (Figure 3A) corresponding to viable cells (annexin V–/PI–, Q3 in Figure 3A), early 13



Page 14 of 37

312

apoptotic cells (annexin V+/PI–, Q4 in Figure 3A) and late apoptotic cells (annexin V+/PI+, Q2 in

313

Figure 3A). Figure 3A shows the gated patterns in double stains of HaCaT keratinocytes with or

314

without curcuminoid pretreatments on hydrogen-peroxide-induced cell death A, and the apoptotic

315

cell populations (annexin V+/PI– and annexin V+/PI+) were quantified, as shown in Figure 3B. After

316

having been treated with 150 µM hydrogen peroxide for 24 h, the apoptotic cells of HaCaT

317

keratinocytes increased from 3.77% to 24.03% and showed significant differences (P < 0.05)

318

compared to the blank (untreated cells). With curcuminoid (1) to (5) pretreatments for 2.5 h (Figure

319

3B), the apoptotic cell populations were 17.6 ± 0.6%, 17.6 ± 0.8%, 18.7 ± 2.1%, 7.4 ± 0.6%, and

320

8.1 ± 1.2%, respectively, which all showed reduced apoptotic cell populations and significant

321

differences compared to those in the control (P < 0.05). Monohydroxy-DMC (4) and

322

monohydroxy-BDMC (5) pretreatments showed the best anti-apoptotic effects.

323

The JC-1 red fluorescence shifted to green, which might imply the early apoptotic phenomena

324

in treated cells.38 Therefore, JC-1 fluorescent dyes were used in the hydrogen-peroxide-induced

325

mitochondrial membrane potential changes in HaCaT keratinocytes with or without curcuminoid

326

pretreatments for 2.5 h before hydrogen peroxide treatments for another 24 h, and expressed as

327

green fluorescence/red fluorescence (Figure 3C). After treatment with 150 µM hydrogen peroxide

328

for 24 h, the ratios of green fluorescence to red fluorescence increased and showed significant

329

differences (P < 0.05) compared to the blank (untreated cells). With curcuminoid pretreatments for

330

2.5 h, only monohydroxy-DMC (4) and monohydroxy-BDMC (5) were shown to reduce the ratios

331

of green fluorescence to red fluorescence, with significant differences compared to those in the

332

control (P < 0.05), which were comparable to those in the blank (untreated cells). These data again

333

revealed that the monohydroxy-DMC (4) and monohydroxy-BDMC (5) showed the best

334

anti-apoptotic effects.

335 14


Page 15 of 37


336

Intracellular ROS Levels. Several reports concern the use of hydrogen peroxide to induce

337

oxidative stress and/or cell death in HaCaT keratinocytes with or without natural compound

338

treatments, which were used to mimic the UV light’s direct and indirect effects on keratinocytes in

339

the skin’s epidermis.30–34 From the results presented in Figure 3B, curcuminoid pretreatments were

340

shown to significantly reduce apoptotic cell populations compared to those in the control (P < 0.05).

341

Therefore, the intracellular ROS levels in HaCaT keratinocytes with or without curcuminoid

342

pretreatments for 2.5 h and then hydrogen peroxide treatments for another 24 h were determined by

343

DFC fluorescence in flow cytometry. The ROS levels in treated cells were plotted as one-parameter

344

histograms with cell count on the y-axis and fluorescence on the x-axis (Figure 4A). Each figure

345

panel contained three overlapping figures, including the repeated untreated keratinocytes (blank)

346

the repeated hydrogen-peroxide-treated keratinocytes (filled gray, control) and the pretreated

347

sample [NAC (the positive control, upper panel, left), curcumin (1) (upper panel, middle), DMC (2)

348

(upper panel, right), BDMC (3) (lower panel, left), monohydroxy-DMC (4) (lower panel, middle),

349

and monohydroxy-BDMC (5) (lower panel, right)]. Each treatment was indicated by an arrow in

350

each figure panel (Figure 4A). The ROS levels in HaCaT keratinocytes (the calculated mean value

351

of fluorescent distributions) in the fixed cell counts were expressed as the fluorescent intensity

352

(Figure 4B). Hydrogen peroxide (150 µM, the control) dramatically increased the intracellular ROS

353

levels and showed significant difference compared to the blank (P < 0.05). The NAC (10 mM)

354

pretreatment was clearly shown to lower the intracellular ROS levels and showed significant

355

difference compared to the control (P < 0.05). With curcuminoid pretreatments for 2.5 h, with the

356

exception of curcumin (1), the other four curcuminoids (2) to (5) were shown to lower the

357

intracellular ROS levels, and showed significant difference compared to the control (P < 0.05);

358

monohydroxy-DMC (4) and monohydroxy-BDMC (5) pretreatments showed the best

359

ROS-lowering effects. The in vitro hydrogen peroxide scavenging activities of curcuminoids were 15



Page 16 of 37

360

performed by the amplex red/HRP system.40 These five curcuminoids showed abilities to directly

361

scavenge hydrogen peroxide and showed no significant difference (P > 0.05) among them (Figure

362

4C). From the results shown in Figure 4, it was clear that monohydroxy-DMC (4) and

363

monohydroxy-BDMC (5) could induce higher levels of ROS-eliminating systems compared to

364

those of curcumin (1), DMC (2), and BDMC (3) in the reduction of intracellular ROS rather than

365

acting as direct radical scavengers.

366 367

Gene and Protein Expressions, and Glutathione Peroxidase Activity. Hydrogen

368

peroxide could activate AP-1 transcription factor in which the c-Fos and c-Jun were the key

369

components.41 Therefore, the relative mRNA expressions of c-fos and c-jun were quantified by

370

qPCR with or without curcuminoid pretreatments before hydrogen peroxide treatments. As clearly

371

shown in Figure 5A, the direct hydrogen peroxide (150 µM, the control) treatments increased the

372

c-fos and c-jun mRNA expressions in HaCaT keratinocytes and showed significant differences

373

compared to the blank (P < 0.05). With curcuminoid pretreatments for 2.5 h, only

374

monohydroxy-DMC (4) and monohydroxy-BDMC (5) pretreatments were shown to lower the c-fos

375

and c-jun mRNA expressions in HaCaT keratinocytes, which were comparable to those of the

376

blank, and showed significant difference compared to the control (P < 0.05).

377

The p53-mediated ROS generation was associated with cell cycle arrest, DNA repair and

378

apoptosis.42, 43 The quantified p53 protein expressions increased after the direct hydrogen peroxide

379

treatments (150 µM, the control), compared to the blank in HaCaT keratinocytes; however, with

380

monohydroxy-DMC (4) and monohydroxy-BDMC (5) pretreatments and then hydrogen peroxide

381

treatments, the quantified p53 protein expressions (Figure 5B) were clearly reduced in HaCaT

382

keratinocytes compared to the control.

16


Page 17 of 37

383


It

was

reported

that

ROS-dependent

HO-1

expressions

could

ameliorate

384

hydrogen-peroxide-induced apoptosis.44 It was found that the HO-1 protein expressions were

385

increased after the direct hydrogen peroxide treatments; however, with monohydroxy-DMC (4) and

386

monohydroxy-BDMC (5) pretreatments and then hydrogen peroxide treatments, the quantified

387

HO-1 protein expressions apparently increased in HaCaT keratinocytes compared to the control

388

(Figure 5C). Figure 5D shows the effects of monohydroxy-DMC (4) and monohydroxy-BDMC (5)

389

treatments

390

monohydroxy-BDMC (5) direct treatments to HaCaT keratinocytes showed increase of GPx

391

activities but not significant (P > 0.05, Figure 5D) compared to the untreated ones. However,

392

monohydroxy-DMC (4) and monohydroxy-BDMC (5) pretreatments and then hydrogen peroxide

393

treatments showed a significant increase of GPx activities in HaCaT keratinocytes (P < 0.05 and P

394

< 0.01, Figure 5E) compared to direct hydrogen peroxide treatments (the control).

on

GPx

activities.

It

was

found

that

the

monohydroxy-DMC

(4)

and

395 396

█ DISCUSSION

397

Hydrogen peroxide was frequently used to induce oxidative stress and/or cell death in HaCaT

398

keratinocytes with or without natural compound treatments that mimicked the UV light’s direct and

399

indirect effects on keratinocytes in the skin’s epidermis.30–34 ROS functioned as the promoter in

400

UV-carcinogenesis and as the inducer of UV-apoptosis.6 The present results revealed that the

401

monohydroxy-DMC (4) and monohydroxy-BDMC (5), minor components of curcuminoids,

402

induced higher levels of intracellular antioxidant defense systems, such as GPx and HO-1, against

403

hydrogen-peroxide-induced oxidative stress and apoptosis of HaCaT keratinocytes. The addition of

404

150 µM hydrogen peroxide for 24-h treatments could reduce HaCaT keratinocyte cell viabilities as

405

shown by MTT stains, and induce cell apoptosis, as confirmed by annexin V-FITC/PI stains, and

406

JC-1 fluorescent changes. Under the non-cytotoxic effects of monohydroxy-DMC (4) and 17



Page 18 of 37

407

monohydroxy-BDMC (5) at 5 µM, HaCaT keratinocytes were pretreated for 2.5 h and then given

408

hydrogen peroxide treatments. The cell viabilities of HaCaT keratinocytes were significantly

409

increased, and the apoptotic cell populations were significantly reduced concurrently with

410

intracellular ROS reductions compared to those treated with hydrogen peroxide.

411

Three major components of curcuminoids, curcumin, DMC, or BDMC, were reported to

412

exhibit in vitro different antioxidant and free radical scavenging activities in literatures. The

413

curcumin, DMC, and BDMC (100 µg/mL corresponding to 272, 283, and 324 µM, respectively)

414

exhibited on anti-linoleic acid peroxidations;45 curcumin (15~45 µg/mL corresponding to 41 to 123

415

µM) showed anti-linoleic acid peroxidations, reducing powers, and scavenging activities against

416

hydrogen peroxide, DPPH radicals, ABTS radicals, superoxide anion radicals, and DMPD

417

radicals;46 the curcumin, DMC, BDMC, and monohydroxy-DMC (10 µM) showed anti-linoleic

418

acid peroxidations;47 curcumin exhibited DPPH, ABTS, galvinoxyl radical scavenging activities,

419

and protective activities against AAPH-induced DNA and erythrocyte damages.20 The five

420

curcuminoids exhibited similar hydrogen peroxide scavenging activity in vitro at the present result.

421

The differences might be either from the concentrations used in literatures generally higher than our

422

present report and/or hydrogen peroxide scavenging activity was only determined in the present

423

result. Several results showed the in vitro antioxidant activities of curcuminoids in which the

424

phenolic OH groups were important in the antioxidant activity, and more hydroxyl groups

425

enhanced the antioxidant effectiveness in vitro.14, 20 In the present results, these five curcuminoids

426

exhibited similar hydrogen-peroxide-scavenging activity (Figure 4C) using the amplex red/HRP

427

system in vitro, and monohydroxy-DMC (4) and monohydroxy-BDMC (5) pretreatments showed

428

the best anti-apoptotic effects, among which were the minor curcuminoids, monohydroxy-DMC (4)

429

and monohydroxy-BDMC (5), with three hydroxyl groups in total; and major curcuminoid

430

components, curcumin (1), DMC (2) and BDMC (3), with a total of two hydroxyl groups. It was 18


Page 19 of 37


431

proposed that monohydroxy-DMC and monohydroxy-BDMC could induce higher levels of

432

antioxidant defense systems than curcumin, DMC or BDMC could against hydrogen

433

peroxide-induced oxidative stress and apoptosis of HaCaT keratinocytes.

434

The GPx is one of the important enzymatic antioxidants (superoxide dismutase, catalase and peroxidase)

responsible

for

ROS

metabolism.44

435

glutathione

436

monohydroxy-DMC (4) and monohydroxy-BDMC (5) direct treatments to HaCaT keratinocytes

437

showed increase of GPx activities but not significant (P > 0.05, Figure 5D) compared to the

438

untreated ones. However, monohydroxy-DMC (4) and monohydroxy-BDMC (5) pretreatments and

439

then hydrogen peroxide treatments showed a significant increase of GPx activities in HaCaT

440

keratinocytes (P < 0.05 and P < 0.01, Figure 5E) compared to direct hydrogen peroxide treatments

441

(the control). The UVA-irradiated HaCaT keratinocytes could induce cytotoxicity via high amounts

442

of hydrogen peroxide generation, and the pretreatment of caffeic acid and ferulic acid could reduce

443

UVA-induced cytotoxicity through upregulation of activities and mRNA expressions of catalase

444

and GPx in the irradiated keratinocytes.48 It was reported that The high level of oxidative and

445

nitrosative stress leads subsequently to induction of GPx mRNA transcription, protein expression.49

446

Therefore, monohydroxy-DMC (4) and monohydroxy-BDMC (5) could upregulate GPx activities

447

under oxidative stress (Figure 5E) rather than acting as direct hydrogen peroxide scavengers. The

448

effects of these two minor curcuminoids on other enzymatic antioxidants, such as catalase and

449

superoxide dismutase, needed further investigation.

It

was

found

that

the

450

Baicalein, a flavone with three hydroxyl groups, but not its glycoside of baicalin, was reported

451

to attenuate hydrogen-peroxide-induced apoptosis via ROS-dependent HO-1 expression in

452

RAW264.7 macrophages.50 Quercetin, but not its glycosides of quercitrin and rutin, was shown to

453

prevent hydrogen-peroxide-induced apoptosis associated with HO-1 expressions in macrophages.51

454

This meant that the aforementioned structure-related flavonoids had different abilities to attenuate 19



Page 20 of 37

455

hydrogen-peroxide-induced apoptosis via HO-1 expressions. The HO-1 catalyzes the degradation

456

of heme to iron, carbon monoxide, and biliverdin. Biliverdin and its metabolized product, bilirubin,

457

were responsible for antioxidant activities in which the HO-1 expression was activated by a range

458

of stimuli, including prooxidants and antioxidants in various cell types.46 The present result

459

revealed that hydrogen peroxide direct treatments could partially activate HO-1 protein expressions

460

in the HaCaT keratinocytes (Figure 5C, lane 2, control), and monohydroxy-DMC (4) and

461

monohydroxy-BDMC (5) pretreatments; hydrogen peroxide treatments could then upregulate HO-1

462

protein expressions that might play roles in lowering ROS in those treated with hydrogen-peroxide.

463

ROS-induced signaling pathways were regulated by a cascade of activation of

464

mitogen-activated protein kinases (MAPKs)50 which in turn activated transcriptional factors, such

465

as p53, NF-κB, AP-1, etc.44, in which the p53 protein served as the cell cycle checkpoint.

466

Inactivation of p53 allowed uncontrolled cell division and genomic instability, and the AP-1 was

467

associated with cell growth and differentiation.42, 44 The p53 is considered as one of the oxidative

468

stress response transcription factors, and ROS acted as both an upstream signal that triggered p53

469

activation and a downstream factor that mediated apoptosis.42 The present results showed that

470

hydrogen-peroxide-induced p53 protein expression (Figure 5B) was concurrent with HaCaT

471

keratinocyte apoptosis, and monohydroxy-DMC (4) and monohydroxy-BDMC (5) pretreatments

472

could downregulate p53 protein expressions, which in turn protected cells against oxidative

473

stress-induced apoptosis. The flavonoids apigenin and luteolin pretreatments suppressed

474

UVA-mediated ROS-induced cell apoptosis of HaCaT keratinocytes via downregulating c-Fos and

475

c-Jun expressions, the key components of AP-1 and MAPKs phosphorylation.40 The gene and/or

476

protein expressions of c-Fos and c-Jun in HaCaT keratinocytes were markedly induced by UVB

477

irradiations,52,53 and pretreatments of SG extracts could dose-dependently suppress both c-fos and

478

c-jun gene expressions.53 20


Page 21 of 37


479

In conclusion, hydrogen peroxide was used to mimic the UV-induced apoptosis of the HaCaT

480

keratinocyte through the elevation of ROS levels. Pretreatments of the minor components of

481

curcuminoids, monohydroxy-DMC (4) and monohydroxy-BDMC (5) were shown to recover the

482

cell viability and reduce apoptotic cell populations through the reduction of intracellular ROS

483

levels via attenuating GPx activities and HO-1 protein expressions and suppressing p53, c-Fos and

484

c-Jun

485

monohydroxy-BDMC as ingredients in the development of antioxidant, anti-radical, or anti-aging

486

cosmetics for skin care needs further investigation.

protein

and/or

gene

expressions.

The

potential

of

monohydroxy-DMC

and

487

488

█ ACKNOWLEDGMENT

489

The authors want to thank Ministry of Science and Technology, Republic of China (NSC

490

101-2313-B-038-002) and Shin Kong Wu Ho-Su Memorial Hospital (SKH-TMU-104-07) for

491

financial supports.

21



492 493 494

Page 22 of 37

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revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 1991, 88,

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in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 1997,

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AP-1-dependent signaling in HaCaT cells. J. Dermatol. Sci. 2011, 61, 23-31.

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stress-induced expression of matrix metalloproteinase-1 by regulating the ERK/JNK/AP-1 pathway

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in human keratinocytes. Biomol. Ther. 2012, 20, 57-61.

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637

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638 639

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Page 29 of 37

640


Figure legends:

641 642

Figure 1. The structures of five curcuminoids used in this study, including curcumin (1),

643

demethoxycurcumin (DMC, 2), bisdemethoxycurcumin (BDMC, 3), monohydroxy-DMC (4), and

644

monohydroxy-BDMC (5).

645 646

Figure 2. Effects of five curcuminoids (A) at 5 µM or (B) at 10 µM on the cell viabilities of

647

HaCaT keratinocytes. The differences of cell viability between curcuminoid treatments and the

648

control group were analyzed using Student’s t-test, and any difference in comparison with the

649

control group was considered statistically significant when P < 0.05 (*), or P < 0.01 (**), or P
0.05

P < 0.01 P < 0.01

P < 0.001

Blank Control

5

P < 0.01 P < 0.01

1

2

Sample (5 µM)

3

4

5

Sample (10 µM)

730 120

731

110

(C)

c

100

732

bc b

733 734 735 736

Cell viability (%)

90 80

a

a

70

a

a

2

3

60 50 40 30 20

737

10 0

738

Blank Control 150 µM H O 2 2

739

1

4

5

Sample (5 µM) + 150 µM H2O2

740

33



741

Page 34 of 37

Figure 3

742 743 744

Blank

Control

(A)

745 746 No.1

No.2

No.3

No.5

No.4

747 748 749 750 751 752

755 756 757

Apoptotic cell populations (%)

754

35

0.7

(B)

30 d

25 c c

20

c

15 b

10 5

ab a

0

758

Green fluorescence/red fluorescence

753

(C) 0.6 b

0.5 b early apopto vs Col 7 - Col 9

b b

0.4 0.3 0.2

a

a

a

0.1 0.0


1

2

3

4

5

Sample (5 µM)+150 µM H2O2


1

2

3

4

5


759 760 761 762 763 764 765 34


Page 35 of 37

766


Figure 4

767 768 769 770 771 772 773 774 775 776 9000

778 779 780 781

(B) ROS levels in HaCaT keratinocytes (fluorescent intensity, RFU)

777

e

e

7500

d c

6000

b

4500

b a

a

3000

1500

0 Blank Control

782

784 785 786 787 788

2

3

4

5


NAC 10 mM+150 µM H2O2

3.5e+5 Hydrogen peroxide scavenhing activity in vitro (fluorescent intensity, RFU)

783

1

150 µM H O 2 2

(C) 3.0e+5

c

b

b

b

b

b

4

5

2.5e+5

4.0e+4

2.0e+4 a

0.0 Blank Control

1

2

3

10 µM H O 2 2

789


35



790

Page 36 of 37

Figure 5

791 3.0

Relative mRNA expression (fold)

792 793 794 795 796 797

c

(A)

c-fos c-jun

bc b

2.5

b

BC

C

B B

1.5 A

a

a A

1.0

a A

0.5 0.0 Blank Control

798

1

150 µM H O 2 2

2

3

4

5


799 800 801 802 803 804 805

808 809 810 811

GPx activity (U/mg protein)

807

250

(D)

(E)

225

100

P>0.05

P0.05

P

Protective Effects of Minor Components of Curcuminoids on Hydrogen

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