In-vitro and in-vivo anti-breast cancer activity of OEO (Oliveria decumbens vent essential oil) through promoting the apoptosis and immunomodulatory effects

Journal Pre-proof In-vitro and in-vivo anti-breast cancer activity of OEO (Oliveria decumbens vent essential oil) through promoting the apoptosis and immunomodulatory effects Tahereh Jamali, Gholamreza Kavoosi, Susan K. Ardestani PII:

S0378-8741(19)32653-4

DOI:

https://doi.org/10.1016/j.jep.2019.112313

Reference:

JEP 112313

To appear in:

Journal of Ethnopharmacology

Received Date: 4 July 2019 Revised Date:

2 October 2019

Accepted Date: 15 October 2019

Please cite this article as: Jamali, T., Kavoosi, G., Ardestani, S.K., In-vitro and in-vivo anti-breast cancer activity of OEO (Oliveria decumbens vent essential oil) through promoting the apoptosis and immunomodulatory effects, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/ j.jep.2019.112313. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

OEO

4T1

In-Vivo

s

Cell Enzymatic Assay without any significant change

Cytokine Assay

Th1 Th2

In-Vitro 2D Culture Cell Prolifiration AnnexinV+/PIROS

Th2 Th1

3D Culture MMp

Casp3 Activity DNA Fragmentation

+ Microscopic Analysis

Tumor Growth Inhibition

Apoptosis

Cell Prolofration

1 2 3

In-vitro and in-vivo anti-breast cancer activity of OEO (Oliveria decumbens vent Essential Oil) through promoting the apoptosis and immunomodulatory effects

4 5

TAHEREH JAMALI1, GHOLAMREZA KAVOOSI2, SUSAN K. ARDESTANI1*

6

1. INSTITUTE OF BIOCHEMISTRY AND BIOPHYSICS, UNIVERSITY OF TEHRAN, TEHRAN, IRAN

7

2. INSTITUTE OF BIOTECHNOLOGY, SHIRAZ UNIVERSITY, SHIRAZ, IRAN

8

* CORRESPONDING AUTHOR: [email protected]

9 10

Abstract:

11

Ethnopharmacological relevance: Oliveria decumbens vent is a valuable plant in Iran, used as a vegetable. Traditionally, the aerial parts of this plant are used to treat the cancer-related symptoms, inflammation, pain, and feverish conditions. However, the scientific evidence related to its traditional effects especially the possible cellular and molecular mechanisms needs to be illuminated. Aim of the study: The main objectives of our study were to explore in-vitro anti-cancer properties of OEO in 2D and 3D conditions, to understand the mechanism of OEO in the induction of death in cancer cells, and to identify in-vivo anti-tumor effect of OEO and induced immunomodulatory effects. Material and methods: OEO was extracted by hydrodistillation and analyzed by GC-MS method. To evaluate the cytotoxic effect of OEO on 4T1 cancer monolayer cells (2D culture) and spheroids (3D cultures), MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was used. Fluorescence staining, various flow cytometry techniques, colorimetric assay, electrophoresis, and comet assay were performed to understand the anti-cancer mechanisms of OEO and determine the death mode in treated 4T1 cells. In animal studies, mouse mammary tumor model was established, the anti-tumor effect of OEO was investigated and ultimately by using the ELISA cytokine assay, immunostimulatory of OEO was studied. Results: According to GC/MS analysis, thymol, carvacrol, p-cymene, and γ-terpinene were identified as main components of OEO. Based on MTT assay, OEO inhibited viability in 4T1 cancer cell without any significant effect on L929 normal cells in 2D, also the anti-proliferative effects of OEO on 4T1 spheroids (3D) was significant but less extent. Our results revealed that OEO induces apoptosis through ROS generation, mitochondrial membrane potential (ΔΨm) disruption, caspase3 activation, and DNA damage. Evaluating the effectiveness of OEO on 4T1 tumor-challenging mice and cytokine assay confirmed anti-tumor effects of OEO and development of an immune response related to Th1 expansion.

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

36 37

Conclusion: These data shed light on the apoptotic mechanisms related to OEO cytotoxicity and introduced this compound as a candidate in cancer therapy.

38 39

Keywords: cancer, Essential oil, spheroids, apoptosis, tumor growth, cytokine

40 41 42 43 44 45 46 47 48 49

Abbrivations: OEO: Oliveria decumbens essential oil. GC/MS: gas chromatography / mass spectrometry. MTT: (3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium Bromide). DCFH2-DA: 2′, 7′- dichlorodihydrofluorescin diacetate. MMp: mitochondrial membrane potential ROS: reactive oxygen species EB: ethidium bromide AO: acridine orange

50

1. Introduction

51

In spite of the development of conventional therapies to combat cancer, including

52

chemotherapy, these therapies cause damage to many tissues and increase the resistance

53

leading to treatment failure. Hence, to minimize these problems, the finding of alternatives of

54

cancer therapy is important, among which herbs have been known as a significant source of

55

novel bioactive compounds for chemotherapeutic development (Roy et al., 2018). Numerous

56

researches in drug discovery from medicinal plants show various pharmacological properties of

57

these plants including anticancer activity. Phytochemical agents obtained from plants work by

58

various mechanisms of action targeting cancer progression and inducing the apoptotic

59

death(Roleira et al., 2015; Roy et al., 2017). Essential oils are natural complexes composed

60

mainly of terpenes in addition to some other non-terpene components. These components

61

exhibit multiple anti-inflammatory, anti-microbial, and anti-oxidant properties and commonly

62

use in pharmaceutical and food industries. An antioxidant is an agent that, when presents at

63

low concentrations, significantly prevents oxidation of a substrate (Carocho et al., 2018). some

64

antioxidant agents may lose their antioxidant activity at high concentrations and therefore act

65

like a pro-oxidant agent (Vertuani et al., 2004). Some essential oils and their components

66

display cancer suppressive activity on a number of human cancer cell lines including breast

67

cancer(Blowman et al., 2018). Apoptosis is the common mode of cell death caused by many

68

anticancer drugs. In apoptotic death, caspase activation plays a central role leading to cleavage

69

of many substrates and ultimately apoptotic events such as DNA fragmentation.

70

Immunotherapy is a relatively modern approach in cancer therapy, by which the host immune

71

system is stimulated to destroy cancer cells. The antitumor efficacy of some chemotherapeutic

72

agents can be achieved through eliciting the immune cells such as Th1 and cytotoxic T

73

lymphocytes, in addition to direct inhibitory effects on cancer cells (Ye et al., 2017). Oliveria

74

decumbens vent. is a traditional medicinal herb belongs to the Apiaceae family and grows in

75

west-southern mountains of Iran. The hydrophobic property of Oliveria essential oil (OEO)

76

allows passing through the membrane and reaching inside the cell. This plant has been used in

77

the traditional treatment of a spectrum of diseases. There are some Persian medical sources

78

that show this plant (also called “Shavasara or Moshk choopan”) has been used as a healing

79

agent for inflammation, cancer, and infections traditionally (Ghahreman and Okhovvat, 2010;

80

Khorasani, 2004; Tonekaboni, 2008). Anti-oxidant, anti-inflammatory and anti-microbial activity

81

of OEO has been investigated in previous studies, however, the anticancer activity of OEO is less

82

clear (Jamali et al., 2018). 4T1 is a mammary carcinoma cell line with highly tumorigenic and

83

invasive properties. Based on these properties, the 4T1 mouse tumor is a suitable animal model

84

for human breast cancer(Pulaski and Ostrand-Rosenberg, 2000). In the current study, our aim

85

was to consider the in-vitro effects of OEO on cell survival and death mode in 4T1 cells in 2D

86

and 3D culture and to study the anti-cancer mechanism induced by OEO in 4T1 monolayers.

87

After that, the anti-tumor activity of OEO was investigated in-vivo in a mouse model followed

88

by the analysis of immunomodulatory effects associated with OEO.

89 90

2. Material and methods

91

2.1. Preparation and analysis of OEO

92

Oliveria decumbense (the local names are “den”, “denak” and “moshkorak”) is the only species

93

of the genus oliveria (the plant name has been checked with https://www.gbif.org and

94

http://www.ipni.org/) from Apiaceae family (herbarium number: 55078). The aerial parts of

95

Oliveria were collected from the mountainous areas of Fars in Iran. For extraction of essential

96

oil, dried plants were subjected to hydrodistillation for 3 h in an all-glass Clevenger-type

97

apparatus (Herbal Exir Co., Mashhad, Iran). In this method, plant materials were soaked in

98

water. The water was boiled to produce steam bearing the most volatile chemicals. The steam

99

was then chilled, and the resulting distillate was collected. The essential oil normally float on

100

top of the aromatic water and was separated from aromatic water via a decanting funnel. The

101

essential oil was dehydrated over anhydrous sodium sulfate and stored at 4 °C.

102

OEO was analyzed by an Agilent 7890A series gas chromatograph (Agilent, Palo Alto, CA, USA)

103

which is equipped with a flame ionization detector (FID) on a fused silica capillary HP-5 column

104

(30 m × 0.32 mm i.d. and film thickness 0.25 µm). The injector volume was 0.1 μl and split ratio

105

of carrier gas (helium) was 1/40. The temperature was set 60°C-240°C. The detector and

106

injector temperatures were set at 250 and 240 °C respectively. GC/MS was carried out by use of

107

Agilent gas chromatograph coupled with Agilent 5975C mass spectrometer equipped with a

108

column HP-5MS. Ionization source temperature was set at 280 °C and the temperature and

109

carrier gas were the same as above. The constituents of OEO were recognized by comparison of

110

Retention indices (calculation for volatile constituents using a homologous series of n-alkanes

111

C8-C25 indices) with those described in the texts (wiley7n.l and NIST05a.L libraries).

112 113

2.2. Preparation of OEO emulsion

114

At first, OEO-water mixture was formed by adding of OEO (1.0 mL) to water (100 mL). Then,

115

polysorbate-20 (100 µg/mL) was added to this mixture. The mixture was incubated (for 24h and

116

at 35°C) and finally, a milky emulsion was formed.

117 118

2.3. 4T1 and L929 cell culture

119

The spontaneously metastasizing murine mammary adenocarcinoma cell line (4T1) and mouse

120

normal fibroblast cell line (L929) were purchased from National Cell Bank of Iran (Pasteur

121

Institute, Iran). L929 and 4T1 cell lines were cultured in RPMI-1640 (Gibco) and DMEM (Gibco)

122

medium respectively. These media were supplemented with 10% FBS (Gibco) and 1%

123

antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Both cell lines were incubated in a

124

humidified incubator (containing 5% CO2 at 37 °C) and passaged using trypsin/EDTA (Gibco) and

125

PBS (phosphate-buffered saline) solution.

126

127

2.4. Formation of spheroids

128

For spheroid formation, a combination of two methods, hanging drop and liquid overlay, was

129

used. The confluent cultures of 4T1 were trypsinized, washed in PBS and resuspended in the

130

medium. Drops (50 μl) containing 3500 cells/ drop were placed on the inner surface of a 60 mm

131

dish lid, which was inverted over dishes containing 10 ml sterilized PBS to humidify the culture

132

chamber. Hanging drop cultures were incubated for 3 days. The formed small spheroids at the

133

bottom of drops were harvested using the wide-mouth tips. Then spheroids were seeded into

134

wells of poly-HEMA–precoated 96-well plates. For the preparation of these plates, 50 μl of poly-

135

HEMA solution (0.5% poly-HEMA (Sigma) in 95% ethanol) were seeded in each well and then air

136

dried was used for 3 days at 37 °C prior to the procedure. In the following, spheroids with

137

homogenous size were seeded into each well of the round bottom of poly-HEMA pre-

138

coated 96-well plate filled by 200 µl complete medium. The plates were incubated in a

139

humidified incubator (at 37°C with 5% CO2). After 3 days, it was taken some photos using invert

140

microscope (Ax overt 25, Zeiss, Germany at 5× and 10× magnitudes) and employed Image J

141

software to estimate the size of spheroids. Formed spheroids were with diameters ranging from

142

500– 600 μm.

143 144

2.5. Cytotoxicity assay in 2D and 3D cell cultures

145

The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay

146

was used to investigate cell viability. Briefly, the 4T1 and L929 cells (1 × 104 cells/ well) were

147

grown in 96-well cell culture plates and after 24h were exposed to various concentrations of

148

OEO (0-200 µg/ml) for 24 h. To study the cytotoxic activity in 4T1 spheroids, after exposure of

149

spheroids to OEO (0-200 µg/ml) for the same duration, spheroids were transferred to 96-well

150

flat bottom plates, supernatant was removed and 200 µl of MTT reagent (5 mg/ml in PBS;

151

Sigma-Aldrich) was added to each well and incubated for 4 h at 37 °C. Subsequently, the MTT

152

solution was removed, and 100 µl of DMSO was replaced in each well and incubated for 30 min.

153

Finally, the intensity of the purple formazan product was measured through the optical density

154

(OD) at 492 nm by using an ELISA reader (Model wave xs2, BioTek, USA). Consequently, IC50

155

(the concentration at which 50% cell proliferation is inhibited) was estimated from the

156

concentration-response curves.

157 158

2.6. Fluorescent staining and apoptosis analysis

159

Apoptosis and cell viability were determined by fluorescent staining with ethidium bromide

160

(EB)/acridine orange (AO). The untreated and treated cells were harvested and centrifuged. The

161

cell pellets were washed with cold PBS and then a mixture of EB/ AO solution (1:1, v/v) was

162

added to the cell suspension at a final concentration of 100 μg/mL. Finally, the stained cells

163

were observed by a fluorescence microscope (Axioskop 2 plus, Zeiss, Germany).

164 165

2.7. Annexin V/ propidium iodide staining and apoptosis analysis

166

Apoptotic 4T1 cells treated with OEO were identified using a FITC (fluoresceinisothiocyanate)

167

labeled Annexin V/propidium iodide (PI) apoptosis detection kit (BioVision). Initially, the cells

168

were treated for 4 h with the IC50 of OEO, then were harvested, washed in cold PBS and stained

169

with PI and FITC-conjugated Annexin V. Finally, stained cells were analyzed using

170

flow cytometry (Partec PAS, Germany).

171 172

2.8. DNA damage analysis using DNA laddering assay

173

4T1 cells were cultured in 60 mm dishes, and harvested after treatment with IC50 doses of OEO

174

and doxorubicin as a positive control. Then based on standard phenol/chloroform extraction

175

procedure, DNA extraction was performed. Extracted DNA was dissolved in TE buffer and

176

loaded on a 2% agarose gel. After staining the gel with EB, DNA was visualized using a gel doc

177

system.

178 179

2.9. DNA damage analysis using Comet Assay

180

DNA damage induction of OEO in 4T1 cells was determined using the alkaline comet assay. 4T1

181

cells cultured in 6 well plates (3 × 105 cells/well) were treated with IC50 concentration of OEO

182

for 4h. The cells were then harvested and centrifuged. Pellets were resuspended in ice-cold PBS

183

buffer. Cell suspensions (1 × 104) were made in 100 µl of 1% low-melting-point agarose (in PBS)

184

and pipetted into a frosted glass slide coated with a thin layer of hot 1% normal-melting-point

185

agarose in PBS, covered with a coverslip, and incubated for 10 min in 4 °C to allow agarose to

186

set. Following removal of the coverslip, the slides were placed in a cold lysis solution (100 mM

187

EDTA, 2.5 M NaCl, 10 mM Tris-base, pH was adjusted to 10 by adding NaOH in 4 °C, and 1%

188

Triton X- 100 added just before use) for 1 h at 4 °C. Then the microscope slides were placed in a

189

chilled electrophoresis buffer (denaturing buffer) (300 mM NaOH, 1 mM EDTA, pH 13) for 30

190

min to allow unwinding of DNA and finally subjected to the electrophoresis for 30 min in 4 °C by

191

applying an electric current of 0.6 v/cm. Then to remove alkali, the slides were washed slowly in

192

neutralization buffer (0.4 M Tris, pH 7.5) for 5 min and then stained with 50μl 1X cyber green

193

and incubated for 15 min in the dark and finally photographed using a fluorescent microscope

194

(Axioscope 2 plus, Zeiss, Germany). At least 100 comets were scored and analyzed using open

195

comet software. Tail length, tail DNA percent, tail moment and olive moment were considered

196

in the comet analysis (George et al., 2018).

197 198

2.10. Reactive oxygen species (ROS) quantitation

199

Intracellular ROS generation was measured using 2′, 7′-dichlorodihydrofluorescin diacetate

200

(DCFH2-DA, Sigma-Aldrich). ROS in the cells causes the oxidation of DCFH and forms the highly

201

fluorescent product 2′, 7′- dichlorofluorescein (DCF). The cells treated with OEO for 12h were

202

harvested, washed with PBS and incubated with DCFH-DA (20 μM) for 15 min in the dark.

203

Finally, the intracellular fluorescence was evaluated using flow cytometry (excitation: 485–495

204

nm; emission: 525–530 nm, FACSCalibur, BD Biosciences).

205 206

2.11. Mitochondrial membrane potential (ΔΨm) assessment

207

Changes in the inner mitochondrial transmembrane potential (ΔΨm) were estimated using

208

Rhodamine123 (Rh123) in 4T1 cells exposed to IC50 of OEO for 12h. Treated and untreated cells

209

were harvested, washed with PBS and incubated in 1 mL of Rh123 (50 µM) at 37 °C for 20 min

210

in the dark. Then the cells were washed twice with PBS to remove extracellular Rh123. Finally,

211

the fluorescent intensity of the Rh123 in the cells was scored immediately by flow cytometry

212

(excitation: 488 nm; emission: 525–530 nm, FACSCalibur, BD Biosciences).

213 214

2.12. Caspase-3 activity assay

215

The caspase activity was measured using caspase-3 colorimetric activity assay kit (BIOMOL

216

International, USA) based on the fluorometric detection of the chromophore p-nitroaniline (p-

217

NA) after the cleavage of the synthetic peptide Ac-DEVD-pNA. Briefly, apoptosis was induced in

218

4T1 cells by different concentrations of OEO. Cells were trypsinized and lysed with the lysis

219

buffer on ice. After centrifugation (12000g for 5 min), the supernatants were collected and their

220

protein value was estimated by the Bradford method. Finally, equal amounts of protein (100

221

µg), 5 µL of caspase-3 substrate (AcDEVD-pNA, 2 mmol/L) and assay buffer were added to each

222

reaction mixture and were incubated for 3 h at 37°C. Finally, caspase-3 activity was calculated

223

at 405 nm with a microplate reader.

224 225

2.13. Animals

226

Female BALB/c mice, 5–6 weeks old and with weights about 19 ± 1.0 g, were purchased from

227

Pasteur Institute, Karaj, Iran and were allowed to adapt to the laboratory conditions for at least

228

1 week before the experiment. All animals were kept under standard housing conditions in

229

polycarbonate cages at 85% relative humidity, 22 ± 3 °C temperature with a 12 hour regular

230

light-dark cycle with standard diet pellets and tap water (Azizi et al., 2017). The experiments

231

were performed with approval from the animal ethics committee of Animal Care and Ethics

232

Committee of the University of Tehran.

233 234

2.14. Animal model and treatment

235

The 4T1 tumor models were generated by injection of 1x106 cells in 50 μl PBS into the back of

236

female BALB/c mice subcutaneously. For the experiment, 35 mice were divided into 7 groups.

237

The first group was a control group without any injection of 4T1 cells, vehicle (sesame oil) and

238

OEO. Group 2 was the control group without any injection of 4T1 cells but with OEO. The third

239

group, negative control, was injected with vehicle alone. Group 4 was kept as tumor control,

240

injected by 4T1 cells and without any treatment. Group 5 was the tumor group treated only by

241

vehicle. Groups 6 and 7 were the treatment groups. Group 6 (major group) was injected by 4T1

242

cells and treated by OEO (intraperitoneal injection, every 2 days for 2 weeks) when the tumor

243

volume reached to 4-5 mm (14 days after tumor inoculation). Group 7 was exposed to the 4T1

244

cell lines and OEO simultaneously to study the prevention role of OEO from tumor incidence. In

245

this study, a high LD50 dose (the dose necessary to produce lethality in 50% of the mice) of 900

246

mg/kg for toxicity was determined and finally, the injected doses were normalized to be 450

247

mg/kg. All mice finally were killed (5 weeks after tumor inoculation), and the spleens were

248

isolated for ex-vivo cytokine production.

249 Table 1. Chemical compositions of Oliveria decumbens essential oil (OEO) identified by retention index and gas chromatography and gas chromatography-mass spectrometry Compounds Retention index Relative percent in OEO a-Thujene

926

0.36

a-Pinene

933

0.31

b-Pinene

976

1.84

Myrcene

990

0.59

n-Decane

999

0.00

a-Phellandrene

1006

0.12

d-3-Carene

1010

0.07

a-Terpinene

1016

0.59

p-Cymene

1026

22.07

Limonene

1027

2.34

b-Phellandrene

1029

0.63

1,8-Cineole

1031

0.14

g-Terpinene

1059

17.80

Terpinolene

1088

0.14

Linalool

1098

0.06

Borneol

1165

0.04

Terpinen-4-ol

1177

0.18

a-Terpineol

1190

0.08

n-Dodecane

1198

0.00

Thymol

1289

25.54

Carvacrol

1296

23.12

Unknown

1302

0.26

Thymol acetate

1354

0.01

a-Copaene

1374

0.01

n-Tetradecane

1398

0.00

Myristicin

1520

3.43

Retention indices were determined using retention times of n-alkanes as standard on fused silica capillary HP-5 column that were injected after essential oil under the same chromatographic conditions.

250 251

2.15. Measurement of Tumor Growth and spleen

252

During the autopsy, tumors were isolated, measured with a caliper. Tumor volumes (mm3)

253

were calculated by the following formula: as the volume = (tumor length) x (tumor width)2/ 2.

254

In addition, spleen and tumors weighed on digital scales(Ren et al., 2018).

255 256

2.16. Cytokine ELISA assay

257

At the end of the study, spleens were removed aseptically and dissected. Single spleen cell

258

suspensions were prepared by gentle homogenization, and red blood cells (RBCs) and red blood

259

cells (RBCs) were disrupted with RBC lysis buffer (20 mM Tris, 160 mM NH4Cl, pH 7.4). The

260

splenocytes were adjusted to 2.5×106 cells/ml in RPMI1640 (Gibco Life Technologies, Germany)

261

supplemented with 10% fetal bovine serum (Invitrogen, Paisley Germany), 100 μg/ml

262

streptomycin, and 100 IU/ml penicillin (Sigma, Germany) 2 mM l-glutamine. Cells were then

263

seeded in 24-well plates and stimulated with 10 µg/ml E. coli LPS (Sigma, L2630) or 10 μg/ml

264

Concanavalin A (Con A, Sigma C7275), at 37°C in a humidified atmosphere of 5% CO2 for 72 h.

265

Cytokine levels (IL-2, IL-4, IL-10, IL-6, IL-1β, TGF-β, TNF-α and IFN-γ) were determined by using

266

ELISA kits (R&D System, Minneapolis, MN). The assays were carried out according to the

267

manufacturers’ instructions. Values were normalized to total protein content as pg of cytokine

268

per mg protein (mean ± SD, n = 5) (Kianmehr et al., 2015).

269 270

2.17. Liver biochemical tests

271

To the assessment of OEO toxicity, peripheral blood was collected into appropriate microtubes

272

and their serums were separated using centrifugation at 3000×g for 25 min. Then the levels of

273

two liver enzymes, serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic

274

pyruvic transaminase (SGPT) of each sample were measured.

275 276

2.18. Statistical Analysis

277

Experimental data processing was carried out using Microsoft Excel 2013 and the GraphPad

278

Prism 5.04 software. The results were presented as the mean ± standard deviation of three or

279

more independent experiments. The significant differences between means were determined

280

by the t-test and one-way ANOVA followed by Turkey’s post-test when statistical significance

281

was P-value ≤ 0.05.

282 283

3. Results

284

3.1. The chemical composition of essential oil

285

OEO was analyzed qualitatively by GC/MS. OEO was found to be rich in phenolic compounds

286

including thymol (25.54%), carvacrol (23.12%), p-cymene (22.07%) and γ-terpinene (17.80%). In

287

general, the monoterpenoid phenols (carvacrol and thymol) form the main components of

288

OEO, but OEO is also composed of monoterpenes (p-cymene and γ-terpinene) (Fig. 1A, Table.

289

1). Since amount of these contents change with some conditions such as collection time,

290

geographical variation, etc., some previous studies reported a different amounts of OEO

291

components, however, in all reports thymol is the main agent (Amin et al., 2005;

292

Hajimehdipoor et al., 2010).

293

294 295 296 297

Fig. 1. A) GC/MS chromatogram of OEO. B) 4T1 Spheroid. Spheroids were imaged by invert microscope. C) % inhibition of proliferation in L929 and 4T1 monolayers (2D) and also 4T1 spheroids (3D) after treatment with increasing concentrations of OEO (for 24 h). To determine IC50 , MTT assay was used. The data are the means±SDs from triplate experiments (P<0.05).

298 299

3.2. Spheroids formation

300

3D cell culture models are better models than 2D cultivation to gain a deep understanding of

301

the biology of cancer. In fact, Multicellular spheroids are mainstay in-vitro models to resemble

302

tumor-like microenvironments and thus use in drug screening. The same anticancer drugs show

303

different effects in 3D and 2D systems so that the cells in 3D cell condition, display a lower

304

sensitivity to some chemotherapeutic drugs compare to 2D culture. To study the effect of OEO

305

on 4T1 cellular spheroids, we used a combination of hanging drop and liquid overlay methods

306

that resulted in spheroid formation. In fact, spheroids were formed and gradually increased in

307

diameter in 5 days. Analysis of phase-contrast images of spheroids indicated that the average

308

diameter of spheroids was calculated 500±5 μm (Fig. 1B).

309 310

3.3. Cytotoxicity induction by OEO in 2D and 3D culture

311

OEO was applied at various concentrations on L929, 4T1 cells, and 4T1 spheroids. MTT assay

312

was performed to detect the anti-proliferative activity of the OEO in these cell lines. In this

313

assay, yellow tetrazolium MTT is reduced in mitochondria to purple formazan by succinate

314

dehydrogenase. Our results showed that OEO does not have any inhibitory effect on L929

315

(IC50>>200) while induces cytotoxicity in 4T1 cells with IC50 of 47.3 μg/ml (Fig. 1C). On

316

spheroids, OEO treatment induced cytotoxicity with IC50 of 130.4 μg/ml (Fig. 1C). Thus,

317

however, the OEO induces its cytotoxic effect in the spheroids, higher IC50 exhibits more

318

resistant of 4T1 spheroids to inhibition by OEO compared to 4T1 monolayer cells. Therefore,

319

these studies demonstrated that OEO is powerful anti-proliferative compounds in 4T1 cells and

320

spheroids, producing concentration-dependent effects.

321 322

3.4. Apoptosis induction by OEO in 4T1 cells

323

Fluorescence microscopic analysis: Using fluorescence microscopy and two dyes of AO and EB,

324

cell death was qualitatively investigated. Ultimately, OEO was found to induce apoptosis in

325

treated cell lines. Indeed, cells stained with AO, are green viable cells, while cells stained with

326

EB are orange late apoptotic cells with condensed or fragmented chromatin and, stained cells

327

with both AO and EB, represent green and orange early apoptotic cells with condensed or

328

fragmented chromatin. Therefore, according to the staining, the chromatin condensation and

329

loss of membrane integrity, it was shown that the mode of cell death was apoptosis in 4T1 cells

330

treated with OEO (for 24h) (Fig. 2A).

331 332 333 334 335 336

Fig. 2. A) Microscopic studies: Fluorescence staining of 4T1 cells a) control cells b) treated cells with OEO (IC50) for 24h. The stained cells (with acridine orange (AO) and ethidium bromide (EB)) were imagined with fluorescence microscopy. B) Annexin-V/PI flow cytometry of 4T1 control and treated cells (with OEO (IC50) for 4h). C) DNA laddering: Lane 1: control sample, Lane 2: the cells treated with OEO (IC50); Lane 3: the cells treated with positive control (IC50) and Lane 4: molecular marker

337 338

Annexin V/PI staining: Flipping of phosphatidylserine (PS) to the outer surface of the cell is one

339

of the properties of apoptosis. The calcium-binding protein of Annexin V binds to PS and thus

340

FITC fluorescence labeled to Annexin V (Annexin V-FITC) can spot PS in apoptotic cells. Since

341

Annexin V is able to contact the ruptured plasma membrane, thus necrotic cell’s membrane is

342

detectable too. Therefore co-staining with PI can be useful to distinguish the apoptotic and

343

necrotic cells. Cells in late apoptosis are positively stained with both Annexin V and PI, in early

344

apoptosis are positively stained with Annexin V, whereas negative staining for both FITC-

345

Annexin-V and PI are recognized as alive cells.

346

Flow cytometry-based detection with Annexin V-FITC/PI can quantify apoptosis in treated 4T1

347

cells. Our findings showed that OEO significantly induces early apoptosis (FITC-Annexin-V+ /PI-)

348

in 4T1 cells (after 4h treatment) (Fig. 2B). The percentage of apoptosis in treated cells compare

349

to that of control cells measured by flow cytometry showed that OEO can be a strong inducer of

350

apoptosis in cancer cells.

351

352 353

354 355 356 357 358

Fig. 3: A) DNA fragmentation by comet assay in a) untreated and b) treated 4T1 cells: DNA migration was observed by fluorescent microscope. Tail DNA percentage, tail moment, tail length and olive tail moment from the comet assay analysis has been exhibited in this fig. B) Caspase 3 activity: in untreated and treated cells (OEO Concentrations: 1/2 IC50, IC50 and 2IC50 µg/ml). The results are based on fold change.

359 360

3.5. DNA fragmentation induced by OEO

361

DNA ladder: Nucleases like CAD (Caspases-activated DNase) degrades DNA during apoptosis.

362

DNA fragmentation is an important hallmark of apoptosis(Zhang and Ming, 2000). Our results

363

showed that treatment of 4T1 with OEO and doxorubicin (an inducer of DNA fragmentation)

364

after 24h led to DNA cleavage and observation of ladder pattern on an agarose gel (Fig.2C).

365

Comet assay: To confirm the ability of OEO in the induction of DNA fragmentation, alkaline

366

comet assay (the single cell gel electrophoresis) was performed(Ganapathy et al., 2016).

367

“Hedgehog tails” which are traces of damaged DNA were observed in the fluorescent

368

microscopy images of 4T1 cells treated with OEO while the control cells possess the intact

369

“heads” without any tails. Therefore, the amount of DNA migration increased after 24h

370

treatment with IC50 of OEO. Collectively, the results indicated that the apoptosis induced by

371

OEO is associated with DNA damage. Tail DNA percentage, tail moment, tail length and olive tail

372

moment from the comet assay analysis were showed in (Fig. 3A).

373 374

3.6. Caspase-3 dependent death induced by OEO

375

Caspase-3 plays a central role in the apoptotic signaling pathway. Caspase-3 cleaves a wide

376

range of the cellular substrates at the C-terminal side of the aspartate residue of the sequence

377

DEVD (Asp-Glu-Val-Asp). The caspase-3 assay is based on the cleavage of Ac-DEVD-pNA as a

378

colorimetric substrate of caspase-3 resulting in the release of the pNA. The absorbance of pNA

379

at 405 nm allowed determination of the several-fold increase in caspase-3 activity in treated

380

cells compared to untreated cells (Fig. 3B). Our findings indicated the apoptotic effect of OEO

381

via a caspase-3 dependent pathway in treated cells.

382 383

3.7. MMP reduction by OEO

384

Since Mitochondrial dysfunction participates in the triggering of apoptosis and is central in the

385

apoptotic pathway, to extra evaluate whether OEO is involved in this mechanism,

386

mitochondrial membrane potential (MMP) in the 4T1 cells was estimated by Rh123 staining

387

using a flow cytometer. Mitochondrial transmembrane potential (ΔΨm) alteration is an

388

indication of the mitochondrial permeability in the cell death systems(Zorova et al., 2018).

389

Indeed, Fluorochrome of Rh123 incorporates into cells dependent on their mitochondrial

390

transmembrane potential. Therefore, reduction of ΔΨm and quenching of Rh123 is along with

391

the mitochondrial membrane permeability. The results showed that in treated cells with OEO,

392

MMP decreased and the percentage of cells with depolarized ΔΨm (increase in Rh123

393

fluorescence means depolarization) increased compared to untreated cells (Fig. 4B). These

394

results confirmed that treatment of 4T1 cells with OEO can induce apoptosis through the

395

mitochondrial pathway. Additionally, since MMP alteration is related to the ROS level,

396

intracellular ROS was considered in the following.

397

398 399 400 401 402 403

Fig. 4: A) Effect of OEO on the reactive oxygen species (ROS) generation in 4T1 untreated and treated cells with OEO (IC50). A right shift of peak demonstrated the increase of ROS in treated 4T1 cells compared to control cells. B) Effect of OEO on mitochondrial membrane potential in 4T1 untreated cells and treated cells with OEO (IC50). A left shift of peak revealed the decrease of Rh-123 fluorescence intensity which is indicator of the loss of MMP.

404

3.8. Intracellular ROS generation by OEO

405

Intracellular ROS plays an important role in the toxicity induced by some antitumor compounds

406

and in the regulation of cellular apoptosis. To deepen into the OEO effects, we determined

407

whether the OEO could modulate the ROS level. ROS generation was evaluated in the treated

408

4T1 cells using DCFH-DA oxidation and forming of fluorescent DCF subjected to flow cytometry.

409

Our results showed that ROS levels in treated 4T1 cells were higher than that in control group

410

(about 7 fold) (Fig. 4A). These results were in compliance with the obtained outcomes in Δψm

411

studies.

412 413

3.9. Tumor growth inhibition by OEO

414

On the day the mice were killed, the mice weight, tumors volume and tumor weight were

415

measured. The results indicated that group 6, which underwent OEO injection, in spite of the

416

higher body weight, displayed lower tumor volume and tumor weight compared to other

417

tumor-bearing control groups (Fig. 5A, B, C). Therefore our results exhibited a significant

418

decreasing effect of OEO on the growth rate of the tumor. Additionally, obtained data from

419

group 7, exposed to 4T1 cell line and OEO simultaneously to study the prevention role of OEO

420

from tumor incidence, showed no effect of OEO on preventing tumorigenesis and also

421

decreasing tumor growth.

422 423

3.10. Spleen weight

424

Spleen weight is associated with tumorigenesis and is an indicator of stress hematopoiesis (Liu

425

et al., 2015). Our results showed a significant effect of 4T1 tumorigenesis on spleen weight. Our

426

analysis indicated that spleen weight was lower in the tumor-bearing group which received

427

OEO (group 6) compared to other tumor-bearing groups (Fig. 5D). This could indicate a

428

decrease in erythropoiesis adopted from the tumor.

429 430

3.11. Enzymatic tests

431

The serum level of SGOT and SGPT was measured. We explored that OEO immunization causes

432

no change in the serum level of these enzymes and therefore has no remarkable effect on liver

433

function (Fig. 5E).

434

435 436 437 438 439 440 441

Figure 5. A) The effect of OEO on tumor growth and spleen in-vivo. B, C) Final tumor weight and volume in 4T1 model groups (p ≤ 0.05). D) Spleen weight index in control and 4T1-bearing groups (p ≤ 0.05). Tumors and Spleens were isolated and weighted, and spleen weight index were calculated as percentage of organ weight (gram, g) per total mouse body weight gram (g). E) The serum level of SGOT and SGPT (No significant difference was found in this measure).

442

3.12. Cytokine determination in spleen cell culture

443

In our study, the cytokine level of splenocytes was measured using a sandwich ELISA assay

444

(R&D System). Elevated levels of proinflammatory cytokine IL-1β, inflammatory mediator IL-6,

445

immunosuppressive cytokine IL-10, and multifunctional cytokine TGF-β and low levels of pro-

446

inflammatory cytokines IL-2, IFN-γ, and TNF-α were detected in untreated and vehicle-treated

447

turmeric mice comparing to control groups. Our results showed that the levels of IL-10, IL-6,

448

TGF-β, and IL-1β reduced in OEO-treated 4T1 inoculated mice (group 6), however, IL-2, IFN-γ,

449

and TNF-α increased in this group compared to other tumor-bearing groups. In addition, our

450

results displayed that IL-4 in splenocytes were not changed significantly in all groups (Fig. 6).

451

Based on these results and the calculation of IFN-γ/IL10 (Fig. 6), the Th1 type of immune

452

response is more likely involved in tumor-bearing mice treated with OEO than other mice.

453 454

4. Discussion:

455

Chemotherapy is considered the most common modality of cancer treatment. A

456

chemotherapeutic should induce cytotoxic effects and reduce tumor growth, with the least

457

damage to normal cells. In spite of extensive efforts for minimization of side effects and

458

enhancement of drug efficacy, due to the inability to achieve the success, chemotherapy

459

application is still very restricted in most of the cancers. Hence, to improve the chemotherapy

460

options, there is a need for alternative agents with low side effects. Todays, plant products are

461

interesting agents in cancer therapy, because of their actions and limited toxicity. These natural

462

components can trigger several signaling pathways and ultimately regulate cell proliferation,

463

apoptosis, etc (Cragg and Newman, 2005). For example, phenolic-rich plants exhibit the high

464

growth inhibition of various cancer cells such as colon, prostate, and cervical cancer cells

465

(Mazewski et al., 2018; Singh et al., 2018).

466

Due to lipophilic properties, the essential oils such as OEO have a unique ability to penetrate

467

the cell membrane and reach inside the cell (Amorati et al., 2013; Gautam et al., 2014). In this

468

study, by analyzing GC/MS and observing the abundance of thymol and carvacrol, OEO was

469

identified as a phenol-rich substance and then its anticancer effects on 4T1 cell line in-vitro and

470

4T1 tumor in-vivo were evaluated. The growth inhibitory effect of OEO on 4T1 cells without any

471

toxicity on L929, obtained from the MTT assay, showed selectivity effect of OEO on cancer cells

472

and strengthened the suitability of OEO as an effective agent.

473 474 475 476

Fig. 6. Measurement of cytokine levels secreted by splenocytes. Spleen cells were isolated and the cytokine levels produced from splenocytes stimulated by LPS or ConA were calculated in different groups (*p<0.05).

477 478

Although the success of monolayer cultivation in the investigation of drugs is significant, 2D is

479

not able to imitate the complexity of clinical tumors. In fact, compacted spheroids from 3D cell

480

culture are a bridge between simple in-vitro 2D culture and the in-vivo tumors.

481

Therefore, in this work, formed 4T1 spheroids were exposed by OEO. Our data obviously

482

confirmed the cytotoxicity of OEO. However, obtaining higher IC50 in spheroid studies showed

483

that OEO has a lower cytotoxicity effect on spheroids compared to 2D monolayer cells. Since

484

cell death induced by most drugs is mediated by triggering the apoptotic pathways, in the

485

following to confirm the apoptotic mode death recognized under a fluorescent microscope,

486

exposure of phosphatidylserine at the cell surface was explored through AnnexinV-(FITC) flow

487

cytometry assay. In addition, DNA fragmentation which is the result of triggering of DNase

488

enzymes by caspase activity was revealed in the treated 4T1 cells with OEO. Detection of

489

caspase3 activity in untreated, treated cells showed the enhancement of caspase3 activity in

490

OEO-treated cells which is essential in apoptosis. Generally, the level of oxidative stress in

491

cancer cells is high. This property can lead to cancer development, however, this level of ROS

492

can cause the susceptibility of cancer cells to additional ROS (Bazhin et al., 2016; Maroof et al.,

493

2012). Apoptosis induced by several anticancer drugs is associated with enhancement of ROS.

494

However, related mechanisms are not clear. In this research, investigation of the effect of OEO

495

on ROS generation showed that treatment of cells with OEO increases ROS level.

496

Although increasing the mitochondria membrane depolarization increases the ROS level, extra

497

ROS also cause to loss of mitochondrial membrane potential (ΔΨm). Indeed, during the early

498

apoptosis, an important property is mitochondrial dysfunction which is a key event leading to

499

caspase activity and finally the intrinsic pathway of apoptosis. Our findings demonstrated that

500

OEO is able to decrease MMP and thus it suggested that OEO induces the apoptotic

501

mitochondrial pathway in OEO-treated 4T1 cell lines.

502

It is worth to mention that in this study, very low concentrations of OEO were used and thus

503

OEO is not able to behave like a pro-oxidant and change the culture medium in the used

504

concentrations. According to previous studies, this essential oil has high antioxidant power and

505

can scavenge the active oxygen and nitrogen radicals. Therefore, OEO not only does not

506

produce active oxygen and nitrogen radicals such as hydrogen peroxide, but it is also capable of

507

eliminating excess radicals in the culture medium (Esmaeili et al., 2018; Karami et al., 2019).

508

Therefore, the effect of OEO on cancer cells is a direct effect resulting from permeability in the

509

cell.

510

To further prove the relevance of our results, the efficacy of OEO on the inhibition of tumor

511

growth in a mouse model of 4T1 breast cancer (in-vivo) was investigated. Our findings based on

512

the reduction of tumor size and weight signified that OEO is effective in decreasing tumor

513

growth. Additionally, in our tumor models, splenomegaly was significantly observed, however

514

in the tumor groups with OEO injection, the spleen weight reduced. This reduction can indicate

515

a decrease in hematopoiesis adopted from the tumor(Hunter Jr and pathology, 2007; Liu et al.,

516

2015). Furthermore, since the exposure to OEO induced no noticeable toxicity regarding animal

517

weight and liver function, OEO could be a promising drug for breast cancer in the future.

518

The immune system plays an important role in tumorigenesis and also cancer therapy. An

519

evaluation of the expression of antitumor cytokines is interesting to confirm the relation

520

between OEO treatment and immune system towards tumor rejection. Therefore, in the

521

present work, we studied the effect of OEO on immune responses of mice-bearing 4T1 breast

522

cancer.

523

Th1 pathways through activating of cytotoxic T lymphocytes, Natural Killer (NK) cells and

524

macrophages can defend against tumors. Indeed, the change of Th1/Th2 balance can influence

525

the direction of the immune response, so that, enhancing the shift from Th2 immune response

526

to Th1 pattern results in anti-tumor immunity (Krohn et al., 2011). TNF-α is a multifunctional

527

cytokine with the important roles in apoptosis, cell survival, inflammation, and immunity. This

528

cytokine is involved in tumor regression while some reports have also explained the role of

529

TNF-α in tumor promotion (Yazdi et al., 2012). IL-2 plays a central regulatory role in the immune

530

system and is necessary for the growth and activity of T cells, NK cells, and macrophages. IFN-γ

531

plays a key role in cancer therapy due to a decrease in tumor growth and also a reduction in the

532

metastasis (Fooladi et al., 2016). In the present study, the TNF-α, IL-2 and IFN-γ measurements

533

as the Th1 cytokines showed a significant increase in the production of these cytokines in OEO-

534

treated tumor-bearing mice compared to other tumor groups.

535

The previous studies also have shown the anti-tumor effects of some other agents attributed to

536

increased IFN-γ and IL-2 and therefore NK activity (Lin and Zhang, 2004; Tsavaris et al., 2002;

537

Yeap et al., 2015). Indeed, NK cells can influence the magnitude of T cell responses, specifically

538

Th1 function through the production of cytokines such as IFN-γ and IL-2 that control the

539

downstream immune responses and finally lead to death and therefore play a major role in the

540

rejection of tumors (DeNardo and Coussens, 2007; Rezvani and Rouce, 2015). Therefore based

541

on high levels of IFN-γ, IL-2 and TNF-α in OEO-treated tumor-bearing mice, we suggested that

542

OEO has anti-tumor effects probably through activating these innate anti-cancer cells.

543

Generally, in cancer patients, the level of Th2 cytokines, IL-4, and IL-10 are high or unchanged

544

(Razali et al., 2016). Our data from the evaluation of IL-4 showed that OEO affects slightly on

545

the production of this cytokine in tumor mice. Numerous studies considered IL-10 as an

546

immune suppressive cytokine which often acts indirectly and obstructs the Th-1 cytokine

547

production (de LeBlanc et al., 2005). High levels of IL-10 decreases cytotoxic T cell responses

548

during tumor growth resulted in inhibition of anti-tumor immunity. However, Dennis et al

549

showed that IL-10 is also essential for Th1 cell function and thus anti-tumor activity (Dennis et

550

al., 2013). In this study, the reduction of IL-10 level, as a Th2 indicator, was obvious in OEO-

551

treated tumor-bearing mice.

552

TGF-β with an anti-inflammatory activity drives the shift toward Th2 responses via IL-10-

553

mediated pathways and directly through inhibition of the Th1-type responses in tumor-

554

challenging mice (Maeda and Shiraishi, 1996). This cytokine is in high levels in tumors

555

environment and contributes to the inhibition of T cells proliferation, causing immune-

556

suppressive and angiogenesis properties. Additionally, Arteaga et al. showed that anti-TGF-β

557

antibodies inhibit the progression of mammary carcinomas and increase mouse spleen NK cell

558

activity (Arteaga et al., 1993). In addition, several cytokines such as TGF-β and IL-10 plays an

559

important role in tumor escape from NK immune surveillance through inhibition of NK cell

560

function (Maroof et al., 2012).

561

Our studies from the investigation of TGF-β production from splenocyte culture showed that

562

OEO significantly decreases TGF-β level in mice-bearing 4T1 cancer and therefore it probably

563

does not block the NK cells activity and T cell proliferation.

564

IL-1 family molecules, especially IL-1β acts with VEGF in maintaining angiogenesis and tumor

565

invasiveness. High IL-1β levels in the tumor microenvironment have been observed in many

566

studies in cancer patients. Our study showed a reduction of IL-1β in OEO-treated tumor mice.

567

Since 4T1 is a cancer with high invasive capacity, this result can lead to a reduction of the

568

chance of tumor invasion (Voronov et al., 2014).

569

Interleukin-6 (IL-6) is a cytokine with a dual role in Th1/Th2 differentiation and therefore

570

tumor-promoting and tumor-inhibitory effects (Diehl and Rincón, 2002) (Knüpfer and Preiß,

571

2007). Sullivan et al. exhibited the effect of IL-6 in the induction of an epithelial-mesenchymal

572

transition phenotype and thus metastasis in breast cancer cells (Sullivan et al., 2009).

573

In our cytokine assay, IL-6 reduced in splenocyte culture of OEO-treated tumor mice compared

574

to other tumor groups. In this study, increased levels of IL-2, INF-γ, and TNF-α but no change in

575

IL-4 level along with low levels of IL-6, IL-10, IL-1β, and TGF-β suggested the expansion of Th1

576

and development of an anti-tumor immune response by OEO. Therefore, taken together, our

577

findings suggest that OEO may play a role in attenuating tumor growth by altering the cytokine

578

milieu and anti-tumoral cell activation during 4T1 cell carcinogenesis.

579 580

5. Conclusion:

581

In this study, we revealed that OEO shows the in-vitro capability to control the proliferation of

582

4T1 breast cancer cell lines in 2D and 3D cultures. Considering the 2D results, this inhibitory

583

effect of OEO is via ROS generation, MMP reduction and finally inducing apoptosis. This

584

outcome was even observed in OEO-treated tumor mice, confirming that OEO in spite of

585

inducing the death in tumor cells, displays the anti-tumor effects due to the development of an

586

anti-tumor immune response. Thus, OEO can be a potential compound with many benefits in

587

breast cancer therapy. However, molecular events and biochemical pathways involved in

588

anticancer activity of OEO are needed to be investigated further in the future.

589 590

6. Acknowledgments

591

This work was supported by the financial support from Shiraz University (grant No. 88-

592

GRAGRST-108). We are appreciated from Dr. Yousef Jamali for graphical designing.

593 594

Competing Interests: The authors declare no competing interests.

595 596

Author contributions:

597

Tahereh Jamali performed the experiments, analyzed data and wrote the main manuscript text. Email

598

address: [email protected]

599

Gholamreza Kavoosi extracted, analyzed, supplied the essential oil and gave some advice in this project.

600

Email address: [email protected]

601

Sussan K. Ardestani supervised the project. Email address: [email protected]

602 603

References:

604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645

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