Mitochondria-Associated Apoptosis in Human ... - ACS Publications

Subscriber access provided by TUFTS UNIV

Article

Mitochondria-Associated Apoptosis in Human Melanoma Cells Induced by Cardanol Monoene from Cashew Nut Shell Liquid Wei-Chao Su, Yu-Feng Lin, Xiang-Ping Yu, Yu-Xia Wang, XiaoDong Lin, Qiao-Zhen Su, Dong-Yan Shen, and Qing-Xi Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01381 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 19, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

1

Mitochondria-Associated Apoptosis in Human Melanoma Cells Induced by

2

Cardanol Monoene from Cashew Nut Shell Liquid

3

(Short title: Cardanol Monoene Induced Melanoma Cells Apoptosis)

4 5

Wei-Chao Su †, #, Yu-Feng Lin †, #, Xiang-Ping Yu†, Yu-Xia Wang†, Xiao-Dong Lin†,

6

Qiao-Zhen Su †, Dong-Yan Shen ‡,*, Qing-Xi Chen †,*

7 8

9

†

State Key Laboratory of Cellular Stress Biology, Key Laboratory of the Ministry of

10

Education for Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University,

11

Xiamen 361102, China ‡

Biobank, The First Affiliated Hospital of Xiamen University, Xiamen 361003, China

15

#

The authors contribute equally in this work.

16

*Corresponding author.

17

E-mail: [email protected] (Qing-Xi Chen), [email protected] (Dong-Yan Shen);

18

Tel/Fax: +86-592-2185487.

19

ABBREVIATIONS USED: CM: cardanol monoene; CNSL: cashew nut shell liquid; DMEM:

20

Dulbecco’s

21

5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC50: half-maximum inhibitory

22

concentration; PI: propidium iodide; AO/EB: acridine orange/ethidium bromide; DMSO:

23

dimethyl sulfoxide; ROS: reactive oxygen species; ΔΨm: mitochondrial membrane potential;

12 13 14

modified

Eagle’s

medium;

FBS:

foetal

bovine

24 1

ACS Paragon Plus Environment

serum;

MTT:

3-(4,


25

ABSTRACT

26

Cardanol monoene (CM) is the major phenolic component extracted from cashew nut

27

shell liquid (CNSL), which has been relevant to wide range of biological effects. In this study,

28

we found that CM could inhibit the M14 human melanoma cells proliferation in a dose

29

dependent and time dependent manner, and the IC50 values were determined to be 23.15 ±

30

2.42 μM and 12.30 ±1.67 μM after 24 h and 48 h treatment, respectively. The flow cytometric

31

analysis demonstrated that CM induced M14 cell cycle arrest at S phase, along with the

32

collapse of mitochondrial membrane potential (ΔΨm) and the accumulation of reactive

33

oxygen species (ROS) level in cells，but the apoptotic cells reduced when treated with

34

Z-VAD-FMK (pan-caspase inhibitor). Western blotting showed that the expressions of p53,

35

cytochrome C, caspase-3 and PARP were up-regulated, the expression level of Bax/Bcl-2

36

ratio increased significantly. The 2527 significant differentially expressed genes were

37

obtained by RNA-seq, which were assigned to 270 KEGG pathways. These results indicated

38

that CM induced M14 cells apoptosis via the ROS triggered mitochondrial-associated

39

pathways, which supports the potential application of CM for the therapy of melanoma

40

cancer.

41 42

KEYWORDS: cardanol monoene, melanoma, apoptosis, mitochondrial dysfunction, ROS,

43

2


Page 2 of 38

Page 3 of 38

44




INTRODUCTION

45

Cashew nut shell liquid (CNSL) is an efficiently available and renewable material.

46

Researchs of recent years showed that CNSL has been widely applied in industrial products,

47

polymerization products and combination with other materials. Cardanols, the phenolic

48

compounds extracted from CNSL, are rich sources of long-chain alkyl substituted salicylic

49

acid and resorcinol1. Cardanol monoene (CM) is one of the three major compounds of

50

cardonals. It has been associated with several of biological effects, owing to its effective free

51

radical scavenging ability. They could act as antioxidant, bactericide, fungicide, insecticide,

52

anti-termite and

53

acetylcholinesterase inhibitors5. Cardanols has been reported to act as an anticancer agent via

54

the inhibition of cell growth against a series of cells lines, such as breast cancer, liver

55

hepatoblastoma, gastric carcinoma and colon adenocarcinoma. 3 However the anti-cancer

56

effect of CM on human melanoma M14 cells has not been reported yet.

molluscicide properties2-4, including

its derivatives designed as

57

Melanoma, the malignancy of pigment-producing cells, is the notoriously aggressive and

58

treatment-resistant form of skin cancer, which causes approximately 75% deaths relevant to

59

skin cancer.6, 7 Therefore, there is a growing interest in drugs that would have the ability to

60

inhibit proliferation and trigger the apoptotic pathway. In our previous study, after purifying

61

from the extracts, we obtained a pure inducer CM and studied have been reported that

62

cardanols could moderate inhibitory activities on tyrosinase effectively.8 However, whether

63

CM could suppress the growth of human melanoma cells and even its mechanism of

64

anticancer action remain unknown.

65

Cell apoptosis induced by most anti-cancer drugs is an effective method for the treatment

66

of malignant cells. Two major pathways to mediate apoptosis are the extrinsic pathway and

67

the intrinsic pathway, also called death receptor pathway and mitochondrial pathway.9

68

Mitochondria, the powerhouse of cells, are the major intracellular organelles that maintain

3



Page 4 of 38

69

homeostasis. Mitochondria have the matrix and and the intermembrane space, which are

70

surrounded by the inner membrane (IM) and the outer membrane (OM) respectively. The

71

disruption of mitochondria is the feature of the programmed cell death. The IM is almost

72

impermeable that allows the respiratory chain to create ΔΨm, the mitochondrial membrane

73

potential. ΔΨm derives from the respiration, electron-transport-chain-mediated pumping of

74

protons out of the IM.10 In the intrinsic pathway, outer stress or signals result in the

75

permeabilization of OM, which leads to the loss of ΔΨm by the decoupling of the respiratory

76

chain. Mitochondria regulates the intrinsic apoptosis pathway that is governed by the proteins

77

of the Bcl-2 family and followed by the activation of caspase-cascades, and forms

78

apoptosome, finally proceeding to apoptosis following the cleavage of PARP proteins, which

79

lead to cell death.11

80

Many cytotoxic agents are identified to have potent anticancer activity for therapy

81

through apoptotic pathway.12 Eucalyptus citriodora resin could inhibit B16F10 proliferation

82

and induce apoptosis for the excessive production of ROS, thus leading to oxidative bursts

83

then the cytotoxicity to B16F10 cells.13 Subamolide E induces A375.S2 cells apoptosis

84

through sub-G1 cell-cycle arrested and caspase-dependent apoptosis.14 Thus that discovering

85

bioactive agents that modulates the mitochondrial functional became an efficient strategy for

86

anticancer therapy. In the this study, we firstly investigated the inhibition effect of CM to M14

87

human melanoma cells and understand its mechanism of action

88 89



90

Chemicals.

91

Cardanol monoene was extracted and purified from CNSL, which was provided by Xiamen

92

Welso Co Ltd., the method described in our recent study (purity 98% of CM).8,

93

chemical structure of CM presented in Figure 1A and structure analysis in Supplementary

MATERIALS & METHODS

4


15

The

Page 5 of 38


94

Figure 1-5 and Supplementary Table1. RIPA buffer, cocktail (protease inhibitor) and

95

DMSO were obtained from Sigma-Aldrich. DMEM, FBS, Trypsin-EDTA, penicillin and

96

streptomycin were purchased from Gibco. Annexin V-FITC/PI apoptosis detection kit,

97

Caspase inhibitor Z-VAD-FMK, PI, MTT, Hoechst 33258, AO/EB and JC-1 were purchased

98

from Sangon Biotech Co., Ltd. (Shanghai, China). Primary antibodies against Bax, MMP-2,

99

MMP-9, Bcl-2, Caspase-3, P53, PARP, cytochrome c, COX IV and Apaf-1 were purchased

100

from ProteinTech Group, Inc., Antibodies against GAPDH were purchased from Sangon

101

Biotech Co., Ltd. Secondary antibody against mouse and rabbit were purchased from

102

Sigma-Aldrich. Other reagents used were to be analytical grade. The water was produced by

103

Milli-Q water purification system.

104

Cell Culture and Treatment.

105

Human M14 cell lines were cultured in DMEM with 10% FBS, and added 100 U/mL

106

penicillin and 100 μg/mL streptomycin, cells grow in a incubator under 37℃ and 5% CO2.

107

Adherent cell were suspended by 1 mL of 1% trypsin-EDTA for 1 min. For the CM treatment,

108

dissolve the CM with DMSO to get certain different concentrations in DMEM.

109

Cell Viability Analysis by MTT Assay.

110

Cell viabilities after treatment with CM were assessed using a MTT assay to evaluate the

111

effect of compound on cell proliferation. Briefly, M14 cells were seeded at the density of

112

7×103 cells/well in 96-well microplates. After 24 h incubating, the medium was changed to

113

the medium in the presence of the CM concentrations (0, 2.5, 5, 10, 20, 30, 40 μM.) in the 200

114

μL of 10% FBS DMEM culture medium. Then the medium was removed for 24 h, after that,

115

add 10 μL of 5 mg/mL MTT and incubated it for 4h, then MTT was replaced with 200 μL

116

DMSO. Finally, the absorbance values was measured at 570 nm by POLARstar Omega

117

automatic multifunctional microplate reader.

118

Cell viabillty (%) = (Asample-Ablank)/(Acontrol-Ablank)×100%

5



119

AO/EB and Hoechst 33258 Staining Assay.

120

M14 cells were seeded in the 6-well plates for 12 h and then treated with the diff erent

121

concentrations of CM (10, 20, 30, 40, 50 μM) for 24 h and 48 h. After treatment, washing

122

cells with PBS and exposed to 10 μg/mL AO/EB at room temperature, followed by

123

observation under the fluorescence microscope (TE2000-U, Nikon, Tokyo, Japan). Another

124

part, staining M14 cells with 10 μg/mL Hoechst 33258 for 15 min at 37°C and morphological

125

changes observed using a fluorescence microscope.

126

Cell Colony Formation Assay.

127

500 cells were seeded in 6 cm plates respectively and divided into two groups. One

128

group for controlwas treated with normal medium, others was incubated with CM (10, 20, 30,

129

40, 50 μM) respectively. Two weeks later, cells were fixed with methanol for 10 min and then

130

stained with Giemsa, PBS washed, air-dried, and then photographed.

131

Cell Migration Assay.

132

In brief, a total of 5×105 M14 cells were seeded onto 6-well plates. A 10 μL pipet tip was

133

used to create an artificial wound area in culture well. After treated with CM or DMSO for 48

134

h, then washed with PBS. The adherent cells were fixed by methanol and stained with crystal

135

violet. The gaps were photographed using inverted phase-contrast microscopy (TE2000-U,

136

Nikon, Tokyo, Japan) equipped with NIS-Elements (Nikon) software.

137

Cell Cycle Analysis by Flow Cytometry.

138

The cell cycle of M14 cells was detected using a flow cytometer, the PI staining method

139

was performed as previously described.16 M14 cells were treated with CM for 24 h, then

140

collecting the adherent and floating cells. M14 cells were fixed in 70% ethanol at -20℃ for 1

141

h after washing with PBS. And resuspended in 200 μL of PBS then added 5 μL of 10 mg/mL

142

RNase and incubated it at 37℃ for 30 min in the incubator. After incubation, DNA was

143

stained with 5 μL of 10 mg/ml PI for 30 min at 4℃ away from light. The DNA content in

6


Page 6 of 38

Page 7 of 38


144

M14 cells were monitored by a FC500 flow cytometer (Beckman Coulter, USA) and the data

145

were performed with ModFit LT 3.3 software.

146

Annexin V-FITC/PI Double Staining for Detecting Apoptosis.

147

M14 cells was detected by the Annexin V-FITC/PI double staining kit (Sangon Biotech,

148

China) and analyzed by a flow cytometry FC500 (Beckman Coulter, USA). In brief, cells

149

seeded in the 6-well plates then treated with different concentrations of CM for 24 h,

150

subsequently trypsinized, and washed with PBS, then suspended the cells using the binding

151

buffer provided by the kit. 5 μL of Annexin V-FITC and 5 μL of PI were added and incubating

152

for 15 min. The proportion of apoptotic cells of each sample was determined by FC 500 flow

153

cytometer, and data was processed and analyzed with FCS software.

154

Determination of ROS Production and ∆Ψm.

155

M14 cells were seeded in 6-well plates for 12 h and treated with CM (10, 20, 30, 40 μM)

156

for 24 h. ROS assay was performed as previously described.17 First, the treated cells were

157

collected, DCFH-DA was added and then cells were suspended and incubated at 37 ℃ for 30

158

min away from light. Then cells were collected to analyze by FC 500 flow cytometer. On the

159

other side, adding JC-1 dyeing liquid to the cellected cells for 1 h. Finally, the ∆Ψm was

160

quantitatively measured and analyzed by a FC500 flow cytometer. Commonly. The proportion

161

of JC-1 fluorescence by red/green reflects the change of ∆Ψm.

162

Western Blotting Assay.

163

The M14 cells were collected and lysed with ice-cold RIPA buffer containing cocktail.

164

The concentration of protein was detected by Bradford method and then heating it at 95℃ for

165

10 min after treated with loading buffer. The same quantity of proteins were seperated by

166

10-15% SDS-PAGE and transferred to PVDF membranes, following the membrane blocked

167

with 5% BSA for 1 h. And then washed with TBST twice for 5 min and incubated with

168

specific primary antibodies at 4℃ for 12 h and secondary antibodies for 1h, respectively. The

7



169

blots were detected with the ECL system (Pierce Co., USA).

170

RNA Extraction, cDNA Library Preparation, and Illumina Sequencing.

171

M14 cells were exposed to the greatest concentration (40 µM) for 24 hours, there were

172

three biological replicates for each concentration. Total RNA was extracted using the TRIzol

173

Reagent (Invitrogen). After the detection of the concentration, quality and integrity of total

174

RNA. mRNA was isolated using oligo-dT beads, following mRNA broke randomly. mRNA

175

was used as templates to synthetize the first-atrand cDNA and therewith synthenizing the

176

double stranded cDNA with response buffer, dNTPs, RNase H and DNA polymerase I.

177

AMPure XP system (Beckman Coulter, Beverly, USA) was used to purify the fragments.

178

Then a cDNA library was created by PCR enrichment. After clustering, the library was

179

sequenced on an Illumina HiSeq 4000 platform to generate 125 bp/150bp paired-end reads at

180

Novogene Co., Ltd., Beijing, China.

181

Quality Assurance and Reads Mapping.

182

The raw data were cleaned by removing reads containing the adaptor sequences, reads

183

containing ploy-N (＞10%) and low quality reads (＞50%) to ensure the high quality of the

184

downstream information analysis. The quality of the clean data was assessed by Q20, Q30

185

and GC content level through Phred score (Qphred = -10log10(e)). After generating a database

186

of splice junctions. Bowtie v2.2.3 and TopHat v2.0.12 were used to build the index of the

187

human reference genome and align the paired-end clean reads respectively.

188

Analysis of Differentially Expressed Genes and Pathway.

189

HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And FPKM

190

(reads per kilo bases per million mapped reads) method was used to calculate the gene

191

expression18. In the this study, DEGSeq R package (1.18.0) and corrected P-value (FDR) ＜

192

0.05 and log2 (Fold change) ≥ 2 were set as the thresholds for identifying significant

193

differential expression genes between control and CM treated cells. Gene Ontology (GO)

8


Page 8 of 38

Page 9 of 38


194

enrichment analysis of differentially expressed genes was implemented by the GOseq R

195

package.

196

enrichment of differential expression genes.

197

qRT-PCR assay

KEGG pathways were exhibited by KOBAS software, which tests the statistical

198

qRT-PCR was performed to verify the result of western blot. After total RNA extracted

199

from M14 cells. RNA was transcribed into cDNA in a 20 μL system according to the

200

Transcript® All-in-one first-stand cDNA Synthesis SuperMix qPCR kit. The real-time PCR

201

was operated on an ABI Prism7000 system (BIO-RAD, USA) using TranStart Top Green

202

qPCR SuperMix kit according to the specification. All the gene expression was using the

203

endogenous control GAPDH as normality. The relative expression level of genes was

204

analyzed with 2−ΔΔCt method. The primers were designed in Table 2.

205

Statistical Analysis

206

All the experiements were repeated three times, all the data are exhibited as the mean ±

207

SD and analyzed with IBM SPSS 19.0 software. The differences between the control and the

208

CM treated were calculated by Student’s t test with GraphPad Prism version 4.0 (GraphPad

209

software, USA).

210 211



212

CM Inhibits Viability and Colony-formation Ability of M14 Cells.

RESULTS

213

To determine the effects of CM on human melanoma M14 cell viability, M14 cell were

214

treated with CM at diverse concentrations for 24 h and 48 h, then the cell viability was

215

assesed by the MTT assay. Just as exhibited in Figure 1B, CM inhibited M14 cells growth in

216

both dose dependent and time dependent significantly. IC50 values are 23.15 ± 2.42 μM after

217

24 h and 12.30 ± 1.67 μM after 48 h of treatment with CM respectively. Such CM caused

218

hardly evident toxicity in human LO2 hepatocytes for comparison. This result of CM having

9



Page 10 of 38

219

no toxicity on normal cells is the same as previous research2. The effect of CM on the

220

colony-formation ability of M14 cells was then investigated. The colony-formation efficiency

221

presented was 96.7% in the control group, but it decreased to be 5.46% to CM treated cells

222

with 40 μM after 7 days (Figure 1C).

223

CM Reduces M14 Cell Migration.

224

Wound healing is a classical method to measure cell migration capability. The cells can

225

sense the existence of a wound and migrate perpendicular to the wound 19. To detect the effect

226

of CM on M14 cells motility, a scratch assay was performed. Wound closure was evaluated 48

227

h through crystal violet staining of M14 cells after treatment. Figure 1D shows that the gap

228

gradually increases with increasing concentration of CM, which indicated that cell migration

229

ability into the scratch decreases after CM treatment. The results of western blot indicated that

230

the expression level of MMP-2 and MMP-9 proteins were down-regulated in a

231

dose-dependent manner distinctly (Figure 3C).

232

CM Induces Cell Cycle Arrest in M14 Cells.

233

To further detect the antiproliferative eff ect of CM on M14 cells, we evaluated the

234

eff ects of CM on cell cycle distribution of M14 cells, which was identified by the DNA

235

content. M14 cells were treated with CM for 24 h, followed by PI staining leads to

236

quantitative DNA staining. Cell cycle distribution is revealed in Figure 2, the percentage of

237

cells in each phase from control, 10, 20, 30, 40 μM were as follows: G0/G1 phase (DNA

238

presynthetic phase), 72.9%, 68.1%, 60.8%, 53.8% and 44.70%; S phase (DNA synthesis

239

phase), 16.1%, 22.2%, 28.0%, 32.4% and 37.9%; G2/M phase (DNA postsynthetic and

240

mitosis phases), 10.9%, 9.7%, 11.1%, 13.8% and 16.9%, respectively (Figure 2B). The cell

241

cycle distribution of M14 cells represented was a dose-dependent manner. The proportion of

242

cells at the S phase increased from 16.1% to 37.9%. The S phase of the cell division cycle is

243

the period where cells replicate their DNA, and thus, the DNA content of S phase is

10


Page 11 of 38


244

intermediate between that of G1 and G2/M cells16. The results were similar to the qPCR

245

results (Figure 2C) where mRNA expression levels of cyclinA1 got decreased and the

246

expression levels of CDK2 got increased. Therefore, CM can inhibit the cell proliferation by

247

arresting the S phase, which resulted in the retardation of cell DNA synthesis. The results

248

indicated that CM could induce M14 cells S phase arrest with a dose-dependent manner.

249

CM Induced Apoptosis in M14 Cells.

250

The flow cytometry analysis was performed to distinguish the living cells and apoptotic

251

cells with Annexin V-FITC/PI double staining. In Figure 3A and 3B, the apoptosis rate of

252

M14 cells triggered by CM was recorded as dose dependent apoptosis features from 0 to 40

253

μM. The proportion of early and late apoptotic cells increased to 43.46% of 40 μM compared

254

to the 1.85% of the control. The Z-VAD-FMK (40 µM) greatly decreased the Cardanol

255

monoene-induced M14 cells apoptosis ratio from 30.56% to 13.17% compared with only

256

Cardanol monoene (30 µM) treatment (Figure 4B, 4C). The results suggested that the

257

inhibition effect on M14 cells results from induction of apoptosis via activation of caspases.

258

To further demonstrate cell apoptosis due to the effect of CM on M14 cells. AO/EB and

259

Hoechst 33258 staining was used to see the change of cell morphologies. AO/EB staining

260

identifies the different stages of cells that are undergoing apoptosis and necrosis. As showed

261

in Figure 3B. M14 cells without CM have a normal morphology and green fluorescence.

262

However, the fluorescence intensity was significantly enhanced when treated with CM and

263

exhibited to be a dose-dependent manner. M14 cells were exposed to CM and stained with

264

Hoechst 33258. Apoptotic cells appeared to show nuclear condensation, brightly stained and

265

fragmentation (Figure 3B), but the apoptotic cells reduced when treated with Z-VAD-FMK

266

(Figure 4A), which presented by the less of nuclear condensation and brightly stained. All of

267

these morphologies are usually found associated with cell apoptosis and were consistent with

268

the result of the double staining. The activated PARP following caspase cleaving is the

11



Page 12 of 38

269

symbol of apoptosis.20 Collectively, the cleavage of PARP, the activation of p53 and caspase-3,

270

cytochrome c release and Apaf-1 (as demonstrated in Figure 3C of western blot results) were

271

increasing in a dose-dependent way. The content of cytochrome c in the mitochondria and

272

cytosol were examined respectively. (Figure 3C) In the presence of CM in the 20, 30 and 40

273

μM doses, the levels of cytochrome c released from mitochondria to cytosol increased in a

274

dose dependent manner. An important role is played by Bax/Bcl-2 to regulate apoptosis,

275

which was just as exhibited in Figure 3D and 3E, where the protein expression level and

276

mRNA expression of Bax, p21 and p53 were elevated while the Bcl-2 was down-regulated

277

synchronously. The results illustrated that CM induced a significant alteration of

278

apoptosis-related protein expression.

279

CM Induces the Generation of ROS in M14 cells.

280

Once intracellular ROS levels become imbalanced, that can cause the mitochondrial

281

dysfunction, which will activate the apoptotic pathway.21 The DCFH-DA assay was used to

282

examine the ROS level of M14 cells. Along with the CM concentration ascension as shown in

283

Figure 5, the mean value of DCF fluorescence value was also gradually elevating, as

284

compared to the control group, the ROS generation in 40 μM group increased by 65.27%

285

which indicated that the ROS levels were increased in a dose dependent way after incubated

286

with the CM. This result suggested that the ROS was the main mediator to induce the M14

287

cells apoptosis.

288

CM Induces Mitochondrial Dysfunction in M14 cells.

289

To assess the polarity of the mitochondrial membrane and involvement of the

290

mitochondrial pathway, we used flow cytometry to examine the quantification of ΔΨm in M14

291

cells upon CM treatment through the mitochondrial-targeted fluorescent probe JC-1. The

292

normal mitochondria of cells showed red JC-1-aggregates in matrix, while the apoptotic cells

293

of JC-1 with the low ΔΨm changed to be JC-1 monomers with green fluorescent. As showed

12


Page 13 of 38


294

in Figure 6, we found that treatment of M14 cells to CM for 24 h led to great decrease

295

red/green fluorescence intensity from 98.56% to 50.37% at 40 μM compared with the control,

296

indicating CM triggered ΔΨm collapse in a dose-dependent way (Figure 6B). It makes clear

297

that CM induced the mitochondrial depolarization in M14 cells.

298

Analysis of DGE Libraries.

299

In order to establish a full quantitative and qualitative gene expression profile of the

300

response of M14 to CM, three DGE libraries of M14 were sequenced after treated with or

301

without CM using Illumina sequencing technology. The quality of three control libraries

302

(Ctrl-1, Ctrl-2, and Ctrl-3) and three CM-treated libraries (CM-1, CM-2, and CM-3) are

303

summarized in Table 1. After eliminating the adaptor sequences and low-quality reads, a total

304

of 26,527,123 and 24,729,689 high-quality reads were obtained from the two groups

305

respectively. Thus, a perfect mapping to the human reference genome was obtained with the

306

averages of 88.62% and 89.04% of reads.

307

Analysis of Differentially Expressed Genes.

308

We used the FPKM method to measure the gene expression levels.18 According to the

309

FPKM values, the genes from Ctrl and CM two groups were presented in (Figure 7A) Venn

310

diagram. And a total of 13,536 (Ctrl) genes and 13,534 (CM) genes were detected. There are

311

12847 genes co-express in Ctrl and CM and the specifically expressed genes number was 689

312

and 687 between the two groups respectively.

313

To identify the differentially expressed genes that show significant change during CM

314

treatment, we detected the genes differentially expressed in the input data from analysis of

315

CM treated read count. And the threshold values were set under corrected P-value (FDR) ＜

316

0.05 and log2 (Fold change) ≥ 2. In this analysis, the DESeq R package was employed.22 As a

317

result, 2527 genes were found to be differentially expressed in the Ctrl and CM groups

318

including up and down regulation of 1365 and 1162 genes respectively (Figure 7B).

13



319

Page 14 of 38

Biological Process Analysis and Pathway Analysis from CM based RNA-seq Data.

320

Clustering analysis is performed to judge the pattern of expression of different genes

321

under varying experimental conditions and the identification the function of unknown genes

322

by clustering the genes through the same or similar expression patterns in different classes.

323

The FPKM of different genes under different experimental conditions was used as the

324

expression level to perform the hierarchical clustering. We clustered the relative gene

325

expression level value log2 (ratios) by H-cluster, K-means and SOM methods, respectively

326

(Figure 7C). The possible molecular function, biological process and cellular component

327

regulated by CM were identified with the software method used in the GO enrichment

328

analysis which is GOseq.23 In the three main categories, the values were 1847, 445 and 1094

329

respectively (Figure 8A).

330

And a large proportion of genes from protein binding (GO:0005515), sequence-specific

331

DNA binding (GO:0043565), protein phosphorylation (GO:0006468), protein kinase activity

332

(GO:0004672) , binding (GO:0005488), organelle lumen (GO:0043233), nucleic acid binding

333

transcription factor activity (GO:0001071), oxidoreductase activity (GO:0016705), cell cycle

334

(GO:0007049), receptor binding (GO:0005102), cytoskeletal protein binding (GO:0008092),

335

regulation of growth (GO:0040008), regulation of cell growth (GO:0001558) and

336

DNA-directed RNA polymerase complex (GO:0000428) were considered great attention. All

337

these data suggest that their overrepresentation after treatment of CM may be due to the cell

338

death of M14 cells.

339

In vitro, pathway significant enrichment was able to determine the most important

340

biochemical and metabolic signal transduction pathways involved in the differential

341

expression genes caused by CM treatment.

342

canonical pathways was done in the KEGG (http://www.genome.jp/kegg/).24 Of the total 2527

343

distinctively expressed genes were allocated to 270 KEGG pathways when cells treated with

The mapping of detected genes to reference

14


Page 15 of 38


344

CM. And most of these distinctively expressed genes were related with Pathways in cancer

345

(83 DEGs), PI3K-Akt signaling pathway (75 DEGs), MAPK signaling pathway (DEGs),

346

Focal adhesion (53 DEGs), Melanoma (21 DEGs), p53 signaling pathway (22 DEGs), Ras

347

signaling pathway (51 DEGs), TNF signaling pathway (30 DEGs) and Rap1 signaling

348

pathway (52 DEGs) (Figure 8B).

349 350



DISCUSSION

351

A great many number of natural bioactive compounds, extracted from various kinds of

352

plants and fruits waste, have been confirmed to be chemotherapeutic agents against various

353

types of cancers, such as fruit shell. CNSL is a renewable available material, extracted from

354

cashew nut shell, is a natural resource with high abundance. CM was the major natural

355

phenolic component extracted and purified from CNSL. In our previous study, it had been

356

reported that cardanol obtained from Thai Apis mellifera propolis induces the apoptosis of

357

BT-474 cells via upregulated p21 protein2 however, there has been no study about the effects

358

of CM on humans melanoma cells or its specific anticancer mechanism at cellular levels.

359

Here on, we investigated whether CM exerts anticancer properties on human M14 melanoma

360

cells and interpreted whether the mechanism of inducing M14 apoptosis relies on a

361

mitochondrial pathway intensively. Data demonstrated the CM inhibited M14 cells

362

proliferation with the IC50 value 23.15 ± 2.42 μM at 24h and 12.30 ± 1.67 μM at 48h. And

363

CM induced the apoptosis notably in a dose dependent and time dependent manner , but the

364

inhibition effect on LO2 human normal liver cells was not obvious (Figure 1B), which was

365

consistent with the findings that is the absence of cytotoxicity of CM to the normal human

366

foreskin fibroblast cell line (Hs27) in vitro, however a great deal of chemotherapy agents used

367

nowadays cause adverse side effects.3

368

To our knowledge, it is the first time to assess the mechanism of CM-induced M14 cell

15



Page 16 of 38

369

apoptosis. Cell death occurs in two forms: apoptosis and necrosis. Namely, apoptosis, a

370

programmed cell death, characterized by morphological changes including cell shrinkage,

371

chromatin condensation, DNA fragmentation and the formation of “apoptotic bodies”. 25 Here,

372

the results revealed that CM has notably inhibited the proliferation of M14 cells in a time and

373

dose dependent manner but not to LO2 human normal liver cells. Meanwhile, the

374

colony-formation ability and cell migration ability were decreasing obviously after CM

375

treatment.

376

Our further study verified whether CM inhibits M14 cells viability via inducing M14 cell

377

cycle arrest and causing apoptosis. In cancer cells, the cell cycle control is deemed an

378

effective method in inhibiting tumor growth.26 Our experiments revealed that CM arrested cell

379

cycle at S phase and prevented entry into G2/M phase in M14 cells by up-regulation of

380

cyclinA protein and down-regulation of CDK2 protein (Figure 2), which were required

381

during the S-G2/M phase of the cell cycle.27, 28 This effect was different from the result that is

382

failure to progress from the G1 to the S phase by cardanol isolated from Thai Apis mellifera

383

propolis.2 These data suggested that cell cycle regulation is a signaling pathway by which CM

384

exerts anti-proliferation effect in M14 cells.

385

The outcomes of Annexin V-FITC/PI staining and flow cytometry analysis demonstrated

386

clearly that the apoptosis ratio of M14 cells triggered by CM has performed dose-dependent

387

features (Figure 3A, 3B). Meanwhile, in order to obtain additional testimony that CM could

388

lead to essential apoptotic features, a sequence of the morphological changes of M14 cells

389

were detected (Figure 3B). Findings like irregular, nuclear condensation, brightly stained,

390

fragmented nuclei and also apoptotic bodies, which usually associate with cell apoptosis, were

391

appeared to be consistent with the outcomes of Annexin V-FITC/PI staining. These significant

392

morphologies were the characteristics of apoptosis, and therefore, the primal discovery

393

revealed that CM induced M14 cell death in an apoptotic way potentially.

16


Page 17 of 38


394

As is known to all, apoptosis is regulated through two pathways, including the intrinsic

395

pathway, also considered as mitochondrial-mediated pathway and another is the extrinsic

396

death receptor-mediated pathway.29, 30 Mitochondrial dysfunction plays a critical role in the

397

intrinsic apoptotic pathways and is the source of signals that initiate apoptotic cell death.31

398

ΔΨm derives from the respiration, electron-transport-chain-mediated pumping of protons out

399

of the inner membrane and is vital for ATP production10. The changes in ΔΨm have been

400

positioned as early, obligate events in the apoptotic signaling. 32 Mitochondrial control of

401

apoptosis was thought to be associated with ATP production, ΔΨm and mitochondrial

402

membrane permeability for the release of certain apoptotic factors.10 In the intrinsic pathway,

403

outer stress or signals result in the permeabilization of outer membrane, which leads in turn to

404

the decoupling of the respiratory chain to loss of ΔΨm and release of apoptosis-related

405

proteins such as cytochrome c into the cytosol. Then cytochrome c combines with Apaf-1 and

406

activates downstream caspases to initiate apoptosis. 33

407

In the recent study, the results of flow cytometry revealed that CM triggered the ΔΨm

408

collapsing (Figure 6). Collapse of ΔΨm leads to mitochondrial structure changed, because of

409

conformational modifications of permeability transition pore. 34,

410

exposing apoptogenic protein cytochrome c from mitochondria to cell cytoplasm, which was

411

initiated by the loss of ΔΨm.

35

This unbalance caused

412

ROS, including superoxides, peroxides, and free radicals, are crucial cellular events and

413

trigger apoptosis through various pathways.36, 37 We checked intracellular ROS and found that

414

ROS were elevating significantly after CM treatment (Figure 5). Studies have reaveled that

415

the accumulation of intracellular ROS was relevant to cell proliferation and apoptosis strongly.

416

Once overabundance of ROS occurred, it would lead to the mitochondrial dysfunction and

417

p53 activation.21 The tumor suppressor gene p53 exhibits a key role in cell apoptosis and has 17



Page 18 of 38

418

close relationship with ROS.38 The results of western blot showed that expression level of p53

419

significantly enhanced when M14 cells exposed to CM (Figure 3C, 3D), which suggests the

420

accumulation and induction of ROS provoked p53 to reach activating M14 cell apoptosis.

421

P53 aims at its transcriptional target Bax and interact with Bcl-2 in mitochondria to

422

induce apoptosis.39 As we all know, in apoptosis Bcl-2 family proteins plays an important role,

423

which are considered to be associated with the liberation of cytochrome c,40 leading to

424

inhibition of the ΔΨm and release of cytochrome c. Bax has been documented to show the

425

pro-apoptotic effect which act as a transcriptional target for p53.41 Bax gave rise to allocation

426

of cytochrome c to cytosol to activate caspase-3.42, 43 It is reported that the expression levels

427

of Bax down-regulated while Bcl-2 was suppressed significantly (Figure 3C, 3D). Therefore,

428

an increase in the Bax/Bcl-2 ratio caused by the exposure of M14 cells to CM after which, the

429

activation of caspase-3 and the cleavage of caspase-3 activates the protein and leads to

430

cleavage of PARP, which leads to cell death ultimately. 44, 45 This result showed that CM

431

regulated M14 apoptosis via modulating Bax and Bcl-2 proteins and caused p53 induction of

432

apoptosis via ROS production.

433

Next research emphasis the inhibition effect of CM in vivo will go on detecting. The

434

xenograft models will be employed to confirm the growth inhibition of M14 tumor in Balb/c

435

nude mice. And then what is the most appropriate effective drug concentration and dosage of

436

CM will be determined, the bioavailability and systemic uptake can be worked out. There are

437

many factors which can affect bioavailability. Bioavailability is closely related to drug

438

efficacy. In vitro, CM inhibited the M14 human melanoma cells proliferation in a dose-and

439

time-dependent fashion, and the IC50 values were 23.15 ± 2.42 μM at 24h and 12.30 ± 1.67

440

μM at 48h. Therefore, the inhibitory activity meets the demand because of its good effect of a

441

micromolar concentration. And the ratio of uptake can be inferred to be more than 50%. It is

442

worth looking forward to expecting that CM can play a role in human tissue without causing

18


Page 19 of 38


443

off target effects. The molecular mechanism can be demonstrated that whether it is consistent

444

between result in vitro and in vivo after treated with CM.

445

In conclusion, CM is a safe, natural and effective compound that could inhibit

446

proliferation of the M14 cells by inducing cell cycle arrest at the S phase and promote M14

447

cells apoptosis in a dose-dependent manner via the ROS triggered mitochondrial-associated

448

pathway. In the future study, the functions of the differentially expressed genes are required to

449

explore a more comprehensive mechanism underlying CM-mediated biological action and

450

evaluate CM in vivo anticancer properties.

451 452

FUNDING

453

This work was financially supported by the Natural Science Foundation of China (No.

454

31570785) and the National Science Foundation for Fostering Talents in Basic Research of

455

the National Natural Science Foundation of China (No. J1310027).

456 457

NOTES

458

The authors declare no competing financial interest.

459

19



Page 20 of 38

460



461

(1) Shobha, S. V.; Ramadoss, C. S.; Ravindranath, B., inhibition of soybean lipoxygenase-1

462

by anacardic acids, cardols, and cardanols. J. Nat Prod. 1994, 57, 1755-1757.

463

(2) Buahorm, S.; Puthong, S.; Palaga, T.; Lirdprapamongkol, K.; Phuwapraisirisan, P.; Svasti,

464

J.; Chanchao, C., Cardanol isolated from Thai Apis mellifera propolis induces cell cycle arrest

465

and apoptosis of BT-474 breast cancer cells via p21 upregulation. Daru. 2015, 23.

466

(3) Teerasripreecha, D.; Phuwapraisirisan, P.; Puthong, S.; Kimura, K.; Okuyama, M.; Mori,

467

H.; Kimura, A.; Chanchao, C., In vitro antiproliferative/cytotoxic activity on cancer cell lines

468

of a cardanol and a cardol enriched from Thai Apis mellifera propolis. Bmc Complem. Altern

469

M. 2012, 12.

470

(4) Kim, D. O.; Lee, K. W.; Lee, H. J.; Lee, C. Y., Vitamin C equivalent antioxidant capacity

471

(VCEAC) of phenolic phytochemicals. J. Agr. Food Chem. 2002, 50, 3713-3717.

472

(5) Nunes Lemes, L. F.; Ramos, G. d. A.; de Oliveira, A. S.; da Silva, F. M. R.; Couto, G. d. C.;

473

Boni, M. d. S.; Guimaraes, M. J. R.; Souza, I. N. O.; Bartolini, M.; Andrisano, V.; do

474

Nascimento Nogueira, P. C.; Silveira, E. R.; Brand, G. D.; Soukup, O.; Korabecny, J.;

475

Romeiro, N. C.; Castro, N. G.; Bolognesi, M. L.; Soares Romeiro, L. A., Cardanol-derived

476

AChE inhibitors: Towards the development of dual binding derivatives for Alzheimer's

477

disease. Eur J. Med Chem. 2016, 108, 687-700.

478

(6) Ko, J. M.; Fisher, D. E., A new era: melanoma genetics and therapeutics. J. Pathol. 2011,

479

223, 241-250.

480

(7) Chatterjee, S. J.; Pandey, S., Chemo-resistant melanoma sensitized by tamoxifen to low

481

dose curcumin treatment through induction of apoptosis and autophagy. Cancer Biol Ther.

482

2011, 11, 216-228.

483

(8) Yu, X. P.; Su, W. C.; Wang, Q.; Zhuang, J. X.; Tong, R. Q.; Chen, Q. X.; Chen, Q. H.,

484

Inhibitory mechanism of cardanols on tyrosinase. Process Biochem. 2016, 51, 2230-2237.

485

(9) Sheridan, J. P.; Marsters, S. A.; Pitti, R. M.; Gurney, A.; Skubatch, M.; Baldwin, D.;

486

Ramakrishnan, L.; Gray, C. L.; Baker, K.; Wood, W. I.; Goddard, A. D.; Godowski, P.;

487

Ashkenazi, A., Control of TRAIL-induced apoptosis by a family of signaling and decoy

488

receptors. Science. 1997, 277, 818-821.

489

(10) Kroemer, G.; Reed, J. C., Mitochondrial control of cell death. Nature Medicine 2000, 6,

REFERENCES

20


Page 21 of 38


490

513-519.

491

(11) Susin, S. A.; Lorenzo, H. K.; Zamzami, N.; Marzo, I.; Snow, B. E.; Brothers, G. M.;

492

Mangion, J.; Jacotot, E.; Costantini, P.; Loeffler, M.; Larochette, N.; Goodlett, D. R.;

493

Aebersold, R.; Siderovski, D. P.; Penninger, J. M.; Kroemer, G., Molecular characterization of

494

mitochondrial apoptosis-inducing factor. Nature. 1999, 397, 441-446.

495

(12) Kepp, O.; Galluzzi, L.; Lipinski, M.; Yuan, J.; Kroemer, G., Cell death assays for drug

496

discovery. Nat Rev Drug Discov. 2011, 10, 221-237.

497

(13) Duh, P. D.; Chen, Z. T.; Lee, S. W.; Lin, T. P.; Wang, Y. T.; Yen, W. J.; Kuo, L. F.; Chu, H.

498

L., Antiproliferative activity and apoptosis induction of eucalyptus citriodora resin and its

499

major bioactive compound in melanoma B16F10 cells. J. Agr. Food Chem. 2012, 60,

500

7866-7872.

501

(14) Wang, H. M.; Chiu, C. C.; Wu, P. F.; Chen, C. Y., Subamolide E from cinnamomum

502

subavenium induces sub-G1 cell-cycle arrest and caspase-dependent apoptosis and reduces

503

the migration ability of human melanoma cells. J. Agr. Food Chem. 2011, 59, 8187-8192.

504

(15) Zhuang, J. X.; Hu, Y. H.; Yang, M. H.; Liu, F. J.; Qiu, L.; Zhou, X. W.; Chen, Q. X.,

505

Irreversible competitive inhibitory kinetics of cardol triene on mushroom tyrosinase. J. Agr.

506

Food Chem. 2010, 58, 12993-12998.

507

(16) Chen, B. H.; Chang, H. W.; Huang, H. M.; Chong, I. W.; Chen, J. S.; Chen, C. Y.; Wang,

508

H. M., (-)-Anonaine induces DNA damage and inhibits growth and migration of human lung

509

carcinoma H1299 cells. J. Agr. Food Chem. 2011, 59, 2284-2290.

510

(17) Chen, Q.; Li, W.; Wan, Y.; Xia, X.; Wu, Q.; Chen, Y.; Lai, Z.; Yu, C.; Li, W., Amplified in

511

breast cancer 1 enhances human cholangiocarcinoma growth and chemoresistance by

512

simultaneous activation of Akt and Nrf2 pathways. Hepatology. 2012, 55, 1820-1829.

513

(18) Mortazavi, A.; Williams, B. A.; McCue, K.; Schaeffer, L.; Wold, B., Mapping and

514

quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008, 5, 621-628.

515

(19) Friedl, P.; Gilmour, D., Collective cell migration in morphogenesis, regeneration and

516

cancer. Nat Rev Mol Cell Bio. 2009, 10, 445-457.

517

(20) Morishima, N.; Nakanishi, K.; Takenouchi, H.; Shibata, T.; Yasuhiko, Y., An endoplasmic

518

reticulum stress-specific caspase cascade in apoptosis - Cytochrome c-independent activation

519

of caspase-9 by caspase-12. J. Biol Chem. 2002, 277, 34287-34294. 21



Page 22 of 38

520

(21) Simon, H. U.; Haj-Yehia, A.; Levi-Schaffer, F., Role of reactive oxygen species (ROS) in

521

apoptosis induction. Apoptosis. 2000, 5, 415-418.

522

(22) Anders, S.; Huber, W., Differential expression analysis for sequence count data. Genome

523

Biol. 2010, 11.

524

(23) Young, M. D.; Wakefield, M. J.; Smyth, G. K.; Oshlack, A., Gene ontology analysis for

525

RNA-seq: accounting for selection bias. Genome Biol. 2010, 11.

526

(24) Kanehisa, M.; Araki, M.; Goto, S.; Hattori, M.; Hirakawa, M.; Itoh, M.; Katayama, T.;

527

Kawashima, S.; Okuda, S.; Tokimatsu, T.; Yamanishi, Y., KEGG for linking genomes to life

528

and the environment. Nucleic Acids Res. 2008, 36, D480-D484.

529

(25) Shi, Y. G., Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell.

530

2002, 9, 459-470.

531

(26) Mork, C. N.; Faller, D. V.; Spanjaard, R. A., A mechanistic approach to anticancer

532

therapy: Targeting the cell cycle with histone deacetylase inhibitors. Curr Pharm Design.

533

2005, 11, 1091-1104.

534

(27) Bertoli, C.; Skotheim, J. M.; de Bruin, R. A. M., Control of cell cycle transcription

535

during G1 and S phases. Nat Rev Mol Cell Bio. 2013, 14, 518-528.

536

(28) Tan, H.; Gao, S.; Zhuang, Y.; Dong, Y.; Guan, W.; Zhang, K.; Xu, J.; Cui, J.,

537

R-Phycoerythrin induces SGC-7901 apoptosis by arresting cell cycle at S phase. Mar drugs.

538

2016, 14.

539

(29) Evan, G., Cancer - A matter of life and cell death. Int J. Cancer. 1997, 71, 709-711.

540

(30) Rego, A. C.; Oliveira, C. R., Mitochondrial dysfunction and reactive oxygen species in

541

excitotoxicity and apoptosis: Implications for the pathogenesis of neurodegenerative diseases.

542

NeurochemE Res. 2003, 28, 1563-1574.

543

(31) Lopez, J.; Tait, S. W. G., Mitochondrial apoptosis: killing cancer using the enemy within.

544

Brit J. Cancer. 2015, 112, 957-962.

545

(32) Zamzami, N.; Marchetti, P.; Castedo, M.; Zanin, C.; Vayssiere, J. L.; Petit, P. X.;

546

Kroemer, G., Reduction in mitochondrial potential constitutes an early irreversible step of

547

programmed lymphocyte death in-vivo. J. Exp Med. 1995, 181, 1661-1672.

548

(33) Yadav, N.; Chandra, D., Mitochondrial and postmitochondrial survival signaling in

549

cancer. Mitochondrion. 2014, 16, 18-25. 22


Page 23 of 38


550

(34) Zhang, Y.; Luo, M.; Xu, W.; Yang, M.; Wang, B.; Gao, J.; Li, Y.; Tao, L., Avermectin

551

confers its cytotoxic effects by inducing DNA damage and mitochondria-associated Apoptosis.

552

J. Agr. Food Chem. 2016, 64, 6895-6902.

553

(35) Hwang, J. H.; Hwang, I. s.; Liu, Q.-H.; Woo, E. R.; Lee, D. G., (+)-Medioresinol leads to

554

intracellular ROS accumulation and mitochondria-mediated apoptotic cell death in Candida

555

albicans. Biochimie. 2012, 94, 1784-1793.

556

(36) Terada, L. S., Specificity in reactive oxidant signaling: think globally, act locally. J. Cell

557

Biol. 2006, 174, 615-623.

558

(37) Martindale, J. L.; Holbrook, N. J., Cellular response to oxidative stress: Signaling for

559

suicide and survival. J. Cell Physiol. 2002, 192, 1-15.

560

(38) Liu, B.; Chen, Y.; Clair, D. K. S., ROS and p53: A versatile partnership. Free Radical Bio.

561

Med. 2008, 44, 1529-1535.

562

(39) Chipuk, J. E.; Kuwana, T.; Bouchier-Hayes, L.; Droin, N. M.; Newmeyer, D.; Schuler,

563

M.; Green, D. R., Direct activation of Bax by p53 mediates mitochondrial membrane

564

permeabilization and apoptosis. Science. 2004, 303, 1010-1014.

565

(40) Gross, A.; McDonnell, J. M.; Korsmeyer, S. J., BCL-2 family members and the

566

mitochondria in apoptosis. Gene Dev. 1999, 13, 1899-1911.

567

(41) Basu, A.; Haldar, S., The relationship between Bcl2, Bax and p53: consequences for cell

568

cycle progression and cell death. Mol Hum Reprod. 1998, 4, 1099-1109.

569

(42) Zhang, H.; Huang, Q. H.; Ke, N.; Matsuyama, S.; Hammock, B.; Godzik, A.; Reed, J. C.,

570

Drosophila pro-apoptotic Bcl-2/Bax homologue reveals evolutionary conservation of cell

571

death mechanisms. J. Biol Chem. 2000, 275, 27303-27306.

572

(43) Antonsson, B.; Montessuit, S.; Sanchez, B.; Martinou, J. C., Bax is present as a high

573

molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J. Biol

574

Chem. 2001, 276, 11615-11623.

575

(44) Yu, F. S.; Yu, C. S.; Chen, J. C.; Yang, J. L.; Lu, H. F.; Chang, S. J.; Lin, M. W.; Chung,

576

J.-G., Tetrandrine induces apoptosis Via caspase-8,-9, and-3 and poly (ADP ribose)

577

polymerase dependent pathways and autophagy through beclin-1/LC3-I, II signaling

578

pathways in human oral cancer HSC-3 cells. Environ Toxicol. 2016, 31, 395-406.

579

(45) Liu, F.; Jiang, N.; Xiao, Z. Y.; Cheng, J. P.; Mei, Y. Z.; Zheng, P.; Wang, L.; Zhang, X. R.; 23



Page 24 of 38

580

Zhou, X. B.; Zhou, W. X.; Zhang, Y. X., Effects of poly (ADP-ribose) polymerase-1 (PARP-1)

581

inhibition on sulfur mustard-induced cutaneous injuries in vitro and in vivo. Peerj 2016, 4.

582 583

24


Page 25 of 38


584

FIGURE CAPTIONS

585

Figure 1. Evaluation of Antiproliferative and antimigratory eff ects of CM at diff erent

586

concentrations on M14 cells. (A) The chemical structure of cardanol monoene. (B) CM effect

587

on cell viability of the M14 cells and LO2 cells tested using MTT assay. The cells were

588

treated with CM (10-40 μM) for 24 h and 48 h. Data are from three independent experiments

589

and presented as percent of the corresponding controls. ∗P ≤ 0.05 and ∗∗P ≤ 0.01. (C)

590

Colony-formation assays were performed in M14 cells treated with CM. (D) Wound healing

591

assay. Images were photographed using a microscope after 48 h incubation.

592 593

Figure 2. Cell cycle distribution after treatment with CM. (A) Cells were treated with CM at

594

various concentrations (0, 10, 20, 30 and 40 μM) for 24 h. Treated and control were stained

595

with PI, changes in cell cycle were examined by the flow cytometry. (B) Histogram shows the

596

percentage of cell cycle distribution in each phases of the cell cycle (G0/G1, S, and G2/M). (C)

597

qPCR analysis for detecting the mRNA levels of cyclinA1 and CDK2. Data are represented as

598

the means ±SD from three independent experiments. ∗P ≤ 0.05 and ∗∗P ≤ 0.01.

599 600

Figure 3. Cell apoptosis induced by CM in M14 cells: (A) Flow cytometric analysis of CM

601

induced apoptosis in M14 cells using Annexin V-FITC/PI. The lower left panel represents the

602

normal cells, the lower right panel represents the early apoptotic cells, the upper left panel

603

right panel represents necrosis cells, the upper right panel represents the late apoptotic cells or

604

undergoing necrotic cells (24 h). Histogram shows the apoptosis cell ratio, which are

605

represented as the means ±SD from three independent experiments. ∗P ≤ 0.05 and ∗∗P ≤ 0.01. 25



Page 26 of 38

606

(B) morphological changes are shown in CM-treated cells by AO/EB and Hoechst 33258, and

607

then observed by fluorescence microscope. (C) Western blot analysis of Bax, Bcl-2, PARP,

608

p53, Apaf-1, caspase-3, MMP-2, MMP-9 in whole cells lysates or cytochrome c in

609

mitochondria and cytosol of M14 cells treated with CM for 24 h. The level of GAPDH was

610

used as a loading control. (D) and (E) qPCR analysis for detecting the mRNA levels of Bax,

611

Bcl-2, Bax/Bcl-2, p53 and p21. Data are represented as the means ± SD from three

612

independent experiments. ∗P ≤ 0.05 and ∗∗P ≤ 0.01.

613 614

Figure 4. Effect of caspase inhibitor Z-VAD-FMK on apoptotic change in M14 cells treated

615

with CM. Cells were treated with 30 μM CM in the presence or absence of 40 μM

616

Z-VAD-FMK. (A) Morphological apoptotic changes in M14 cells. The nuclei were stained

617

with Hoechst 33258 and visualized under a fluorescence microscope. (B) Flow cytometric

618

analysis of apoptosis using Annexin V-FITC/PI (C) The Histogram analysis of apoptosis cell

619

ratio.

620

621

Figure 5. CM increases intracellular ROS levels in M14 cells. M14 cells were incubated with

622

CM at various concentrations (0, 10, 20, 30 and 40 μM) for 24 h. The changes of ROS level

623

were examined by the flow cytometry, and presented as histogram. Data are the three

624

independent experiments. ∗P ≤ 0.05 and ∗∗P ≤ 0.01.

625

626

Figure 6. CM induced ΔΨm collapse on the M14 cells. (A) Cells were treated with CM at

627

various concentrations (0, 10, 20, 30 and 40 μM) for 24 h. Changes in ΔΨm were examined

628

by the flow cytometry. (B) Histogram shows the percentage of JC-1 monomer ratio which

26


Page 27 of 38


629

means the percentage of mitochondrial depolarization. Data are the three independent

630

experiments. ∗P ≤ 0.05 and ∗∗P ≤ 0.01.

631 632

Figure 7. Analysis of differentially expressed genes. (A) Venn diagram showing the genes

633

expressed in the control and CM-treated samples. Among these genes, 13,534 are expressed in

634

Ctrl and CM samples, 12847 are co-expressed. The number of specifically expressed genes

635

between the two groups is 689 and 687 respectively. (B) Expression level and statistics

636

analysis of the DEGs between samples. Between the Ctrl and CM samples libraries, there are

637

1365 genes up-regulated and 1162 genes down-regulated. (C) Hierarchical cluster analysis of

638

gene expression based on log ratio FPKM data. The colour means Log2 (Fold-change) of the

639

differential expression profiles. Green represents the genes of lower expression, red represents

640

the genes of high expression, columns represent individual experiments, and rows were

641

transcriptional units.

642 643

Figure 8. Biological process analysis and pathway analysis during CM treatment. (A) the

644

histogram of GO classification, including the down-regulated (A2) and up-regulated (A3).

645

The y-axis indicates the number of genes in a category. In three main categories of GO

646

classification, there are 12, 8 and 10 functional groups in CM treatment. Among these groups,

647

the GO terms binding (GO: 0005488) in the molecular function categories was dominant in

648

the treatment of CM. (B) Scatter plot of KEGG pathway enrichment statistics and the most

649

enrichment pathway during the treatment of CM. (B1) Top 20 statistics of pathway

650

enrichment after CM treatment. (B2) Top 20 statistics of pathway (down-regulated)

651

enrichment after CM treatment. (B3) Top 20 statistics of pathway (up-regulated) enrichment

652

after CM treatment.

27



Page 28 of 38

TABLES Table 1. Summary of DGE Sequences analysis. Sample

Clean

Total

Raw reads name

reads

mapped

Q20(%)

Q30(%)

GC content(%)

Ctrl-1

25061064

24469754

21660207

97.7

94.5

49.7

Ctrl-2

32333914

31574430

27993410

97.7

94.4

49.5

Ctrl-3

24106154

23537184

20873301

97.7

94.6

49.7

CM-1

26942398

26191580

23371886

98.0

95.2

49.8

CM-2

23437344

22789354

20287367

97.9

94.9

49.3

CM-3

25952928

25208132

22399937

97.9

95.0

49.7

28


Page 29 of 38


Table 2. Primers used in gene expression analyses. Gene

Primer forward (5’-3’)

Primer reverse (5’-3’)

GAPDH

CAGGAGGCATTGCTGATGAT

GAAGGCTGGGGCTCATTT

Bax

TCTGACGGCAACTTCAACTG

TTGAGGAGTCTCACCCAACC

Bcl-2

CCTGGTGGACAACATCGCC

AATCAAACAGAGGCCGCATGC

P21

GTCAACTGTCTTGTACCCTTGTG

CGGCGTTTGGAGTGGTAGAAA

P53

GGAAATCTCACCCCATCCCA

CAGTAAGCCAAGATCACGCC

CyclinA

TCCATGTCAGTGCTGAGAGGA

GAAGGTCCATGAGACAGGC

CDK2

ACCTCCAGGAGATTCCAGACC

CCCAGGTTTGAGAGCAGTTCC

29



FIGURES Figure 1.

30


Page 30 of 38

Page 31 of 38


Figure 2.

31



Figure 3.

32


Page 32 of 38

Page 33 of 38


Figure 4.

33



Figure 5.

34


Page 34 of 38

Page 35 of 38


Figure 6.

35



Figure 7.

36


Page 36 of 38

Page 37 of 38


Figure 8.

37



TABLE OF CONTENTS GRAPHIC

38


Page 38 of 38

Mitochondria-Associated Apoptosis in Human ... - ACS Publications

Recommend Documents