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10-Gingerol, a Phytochemical Derivative from Tongling White Ginger, Inhibits Cervical Cancer: Insights into the Molecular Mechanism and Inhibitory Targets Fang Zhang, KIRAN THAKUR, Fei Hu, Jian-Guo Zhang, and Zhao-Jun Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00095 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Journal of Agricultural and Food Chemistry

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Title and authorship

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10-Gingerol, a Phytochemical Derivative from Tongling

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White Ginger, Inhibits Cervical Cancer: Insights into the

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Molecular Mechanism and Inhibitory Targets

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Fang Zhang †, Kiran Thakur †, Fei Hu †, Jian-Guo Zhang †, Zhao-Jun Wei † ‡ *

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†

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230009, People’s Republic of China

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‡

School of Food Science and Engineering, Hefei University of Technology, Hefei

Agricultural and forestry specialty food processing industry technological innovation

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strategic alliance of Anhui province, Hefei 230009, People’s Republic of China

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Fang Zhang [email protected]

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Kiran Thakur [email protected]

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Fei Hu hufei@ hfut.edu.cn

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Jian-Guo Zhang [email protected]

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*Correspondence: E-mail: [email protected]; Tel: +86-551-62901539; Fax:

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+86-551-62901539.

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ACS Paragon Plus Environment


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Abstract: With an aim to evaluate anti-cancerous activities of 10-gingerol (10-G)

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against Hela cells, it was purified and identified from Tongling White Ginger by

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HSCCC, UPLC-TOF-MS/MS and NMR analysis, respectively. 10-G inhibited the

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proliferation of HeLa cells at IC50 (29.19 µM) and IC80 (50.87 µM) with altered cell

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morphology, increased cytotoxicity and arrested cell cycle in G0/G1-phase. The most

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cell cycle related genes and proteins expression significantly decreased, followed by

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slight decrease in few and without affecting cyclin B1 and cyclin E1 (protein). Both

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death receptors significantly upregulated and activated apoptosis indicators (caspase

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family). Furthermore, significant changes in mitochondria dependent pathway

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markers were observed and led to cell death. 10-G led to PI3K/AKT inhibition and

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AMPK activation to induce mTOR mediated cell apoptosis in Hela cells. Our results

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can be an asset to exploit 10-G with other medicinal plant derivatives for future

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applications.

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Keywords: Tongling White Ginger; 10-Gingerol; Hela cells; Apoptosis;

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PI3K/AKT/AMPK/mTOR pathways

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Introduction

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‘Tongling White Ginger’ in the ideal climatic conditions of Tongling (Anhui province,

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China) after cultivation for thousands of years enjoys a reputation on the characteristic

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thin white peel, tender flesh, rich in juice and flavor characters, also traditionally

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regarded as one of the top gingers in China.1 In general, ginger is widely consumed as

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a popular spice throughout the world, and had been cultivated for traditional oriental

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medicinal usages in China, India, Japan and other Asian countries.2, 3 Pharmacological

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investigations have revealed the chemo-preventive and chemotherapeutic effects of

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ginger and its major pungent ingredients on variety of cancer cell lines and animal

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models.4 Previous studies have shown that the extracts of ginger possess variety of

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biological and pharmacological activities, including antioxidant,5 anti-inflammatory,6

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anti-cancer,7 glucose lowering,8 musculoskeletal disorder,9 osteoarthritis,10 migraine11

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and cardiovascular protective activities.12

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Gingerols have been reported as main functional components from ginger, which

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share the same vanillyl moiety and possess a similar chemical structure with a

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different unbranched alkyl carbon chain.13 Based on alkyl side chain lengths,

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gingerols are assigned as 4, 6, 8, 10, 12-gingerol and so on.14 Previous report

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suggested that phytochemicals extracted from ginger are better studied for their

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anti-cancerous attributes.15 Similarly, gingerols have been reported to exhibit

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anti-oxidant, analgesic, anti-pyretic, anti-inflammatory, anti-neuroinflammatory16 and

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anti-tumorigenic activities.17 As the major pharmacologically active member of

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gingerols, 10-G had attracted many attentions and it was reported to exhibit 3



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anti-cancerous,18 anti-neuroinflammatory,16 anti-oxidant and anti-inflammatory13

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attributes. However, the detailed molecular mechanism of its anti-cancerous activities

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is still in its infancy.

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The series of protein kinase complexes (cyclin-dependent kinases (CDKs) and their

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activating partners) regulate cell cycle progression in eukaryotic cells.19 The cyclin

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A/E - CDK-2 and cyclin D1 - CDK-4/6 complexes play vital role in regulating the

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G0/G1 checkpoint. Apoptosis, a programmed cell death involves several

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morphological changes and cellular signaling pathways.20 Among them, intrinsic

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(mitochondrial related which involves Bcl-2 family members, release of cytochrome c

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and activation of series of caspases) and extrinsic (death receptor) pathways are

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important.21, 22 Moreover, PI3K/AKT pathway is important in preventing cells from

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undergoing apoptosis and leading to the pathogenesis of malignancy. Besides, it is

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also associated with the regulation of cell cycle progression.17

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Drugs used in classical chemotherapy are expensive, not target specific and lead to

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severe systemic side effects.23 Multiple uses of these drugs help the body to develop

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resistance due to heterogeneity of cell types and clonal selection. Here comes the role

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of traditional dietary ingredients from plants, besides their safety and easy availability

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and multiple targets/pathways sites, they are consumed as part of daily diet. Hence,

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our approach is promising which recommend administration of dietary

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phytochemicals that possess biological active components for inhibition of cancer

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cells. However, applications of plant components for cancer treatment/prevention

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require better understanding of anticancer functions and elucidation of their 4


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mechanisms of action in depths. Despite evidences for the number of biological

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effects of 10-G as the second major component of gingerols, its anti-cancerous effects

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related to apoptosis have not been fully explored till date.18

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This study has reported the separation and identification of 10-G from Tongling White

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Ginger, and evaluation of its anti-cancerous activities against HeLa cells. The effects

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of 10-G on the cell proliferation, morphology, cell cycle, and apoptosis were

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investigated. Furthermore, cell cycle and apoptosis related genes and their proteins

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were studied by RT-qPCR and western blot, respectively. The anti-cancerous activities

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of 10-G on the cell proliferation, cytotoxicity, morphology, cell cycle, and apoptosis

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would extend the utilization of ginger as functional foods in the future.

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Materials and methods

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Materials

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Chinese white ginger rhizomes (Zingiber officinale) were procured from Tongling

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White Ginger Development Co., Ltd. All chemicals used in the study were of

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analytical grade. All tissue culture reagents, e.g., Dulbecco’s Minimum Essential

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Medium (DMEM), Roswell Park Memorial Institute (RPMI-1640), fetal bovine

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serum, trypsin solution without EDTA, penicillin and streptomycin were purchased

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from Invitrogen (Carlsbad, USA). Pifithrin-µ, Wortmannin, Deguelin, WZ4003 and

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Temsirolimus were obtained from Abmole Bioscience Inc. (USA).

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Preparation of crude extraction

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Fresh ginger rhizomes were sliced, grinded (60 meshes) and vacuum-dried at 50°C, 5



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and then addition of α-amylase at 0.5% (IU) concentration, followed by pH to 5.0 ±

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0.2 which was adjusted with citric acid. Mixed aqueous solution was incubated at

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50°C for 120 min for collection of enzyme-treated powders. The residue was mixed

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with 40 mL 90% methanol in an ultrasonic bath at 45°C for 1 h to extract gingerols.

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After centrifugation, the supernatant was evaporated under reduced pressure at 40°C

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to remove the methanol, and was freeze-dried for further use.24 Gingers used for

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experiment were from the same batch.

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Purification of 10-gingerol

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10-G was purified from crude extraction according to the procedure of HSCCC

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separation by Zhan et al.25 with some modification. In present study, the HSCCC

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system was carried out using binary solvent system which consisted of

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n-hexane-chloroform-methanol-water (2:5.5:6.5:1, v/v/v/v) system for the isolation of

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10-G. 100.0 mg of sample in 5 ml solvent mixtures of lower phase and upper phase

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(1:1, v/v) was added. After the mixed solvents were thoroughly equilibrated at room

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temperature, the upper stationary phase at a flow rate of 10 mL/min was pumped into

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multilayer coiled column until fully filled with the fluid, and the apparatus was rotated

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at 800 rpm. Keeping the volume of the stationary phase under the reach of

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equilibration, the lower mobile phase was eluted in a descending mode at a flow rate

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of 1.3-2.0 mL/min followed by immediate injection of the samples. The effluent was

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continuously monitored with a UV detector at 280 nm to obtain chromatograms and

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the fraction of peak fraction was collected.

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HPLC analysis of ginger extraction

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HPLC analysis was performed on a Shimadzu LC-8A system, equipped with 515

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HPLC pump, 20 µL injection loop and SPD-m10AVP UV detector with system

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controller SCL-10AVP. HPLC analysis was performed according to the description by

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Pawar et al.26 The sample was identified with a reverse phase Alltech-C18 column

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with 5 µm particle size. The mixture solution of acetonitrile: water (75:25) were used

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as the mobile phase at a flow rate of 0.8 mL/min for chromatographic separation of

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10-G. The sample of ginger extraction was dissolved in methanol for detection with

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the wavelength under 280 nm, and the injection volume was 20 µL per sample which

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was executed for 20 min of each procedure.

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UPLC-TOF-MS/MS analysis of ginger extraction

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The ginger extraction dissolved in methanol (HPLC grade) and was analyzed by

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Waters ACQUITY UPLC system (Waters, Milford, MA, USA) and LCT Premier XE

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time-of-flight (TOF) mass spectrometer (Waters, Milford, MA, USA) equipped with

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vacuum degasser, binary pump, auto-sampler, column compartment, and diode array

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detector. ACQUITY UPLC BEH-C18 column (5 cm, 2.1 mm, 1.7 µm) was used for

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chromatographic analysis by ACQUITY UPLC BEH-C18 column (5 cm, 2.1 mm, 1.7

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µm) at a flow rate of 0.3 mL/min at 30°C. A binary gradient elution system composed

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of eluent A (deionized water) and eluent B (acetonitrile) was applied as followed27:

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maintaining 70% of A and 30% of B at the beginning 3 min, and then the content of B

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was increased from 30% to 70% at 3.2 min and maintained to 3.5 min. The injection

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volume was set at 10 µL, and each run was followed by equilibration time of 5 min.

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And the elution system was supplied according to same system given for 10-G

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detection. The MS incorporated with electrospray ionization (ESI) interfaces. The

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positive and negative ion polarity mode was set for the ESI source with the voltage on

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the ESI interface maintained at approximately 5.5 kV. The conditions were set as

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followed: de-solvation gas flow at 600 L/h at 350°C, cone gas flow at 60 L/h and

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source temperature at 110°C, collision energy 10.0/35 V, nebulizer pressure 50 psi and

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scan range m/z 100-1000.

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Nuclear Magnetic Resonance

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1

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MHz). Chemical shifts in parts per million (in ppm) of the 1H-NMR spectra were

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referenced to tetramethylsilane (δ = 0 ppm) in CDCl3 as an internal standard. And the

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13

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Cell culture

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Hela cell and HEK293 cell lines were obtained from Shanghai wei atlas biological

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technology co., LTD. The Hela cells was grown in RPMI-1640 medium supplemented

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with 10% fetal bovine serum (FBS) and 100 µg/mL streptomycin and 100 U/ml

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penicillin G at 37°C in a humidified atmosphere of 5% CO2.28 The Human embryonic

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kidney HEK293 cells was cultured in Dulbecco minimum essential medium (DMEM)

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containing FBS (10%), streptomycin and penicillin G at 37°C in a CO2-incubator. The

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culture medium was revived twice in a week and detached by trypsin, and then plated

H-NMR spectra and 13C-NMR spectra were recorded with Bruker AVIII-600 (600

C-NMR spectra were calibrated with CDCl3 (δ = 77.00 ppm).

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in 6- or 96-well plates before experiments. To evaluate the effect of inhibitors

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targeting signal transduction pathway on 10-G induced apoptosis in Hela cells, the

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cells were pre-treated with respective experimental conditions as per protocol for each

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inhibitor.

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Determination of cancer cell inhibition

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The cell proliferation was assayed according to the manufacturer’s instructions of the

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Cell Counting kit-8 assay kit (DOJINDO Corp.). Cells were dispensed at the initial

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density of 2 × 105 cells per well of a 96-well microplate with the volume of 100 µL

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and pre-incubated at 37°C. After 10 hours, medium was replaced with RPMI-1640 or

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DMEM 10% FBS containing different concentrations of 10-G (5-120 µM) or

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5-fluorouracil (5-FU, 50 µM) solution for Hela or HEK293 cells, respectively. And

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then the cells were further continuously cultivated for 48 h. Number of viable cells

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were monitored using WST tetrazolium salt (CCK-8) followed by method Johnson et

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al.29 Inhibition ability was expressed as the percentage of absorbance in the treated

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cells compared to negative control:

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Cell inhibition ability (%) = (ODnegative control - ODtreatment)/(ODstandard - ODblank)×100%.

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All the experiments were performed in triplicate. The inhibition rate of 10-G on the

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cell lines can be evaluated as follow: y =

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A1 - A 2 1+e

( x − x 0 ) / dx

+ A2

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Where y is the inhibition rate of 10-Gingerol at concentration (x), A1 and A2 are the

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maximum and initial inhibition value, respectively. X0 is the concentration required to

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reach half of the maximum inhibition intensity, and dx is the apparent first-order 9



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aggregation constant.

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Effect of 10-gingerol on cytotoxicity assessment

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Hela cells and HEK293 cells were cultured at the density of 2 × 105 cells per well in

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96-well plates for 10 h, and were replaced into the medium including 10-G at

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different concentration or 5-FU solution (65 µM). After 48 h, the culture medium was

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collected and LDH cytotoxicity was determined by a microplate reader at 490 nm

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using the cytotoxicity LDH assay kit (Dojindo).30 All experiments were repeated three

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times.

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Effect of 10-gingerol on cancer cell cycle

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Cell cycle distribution was detected by the classical propidium iodide (PI) staining

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method and flow cytometric analysis. 1×106 cells were fixed in cold 70%ethanol,

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RNase treated, and stained with propidium iodide (BD) followed the manufacturer’s

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instructions Cell Cycle Analysis kit (BD Biosciences). Cells were analyzed for DNA

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content by Flow Cytometry (BD FACSCalibur, USA).31 All the experiments were

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performed in triplicates and expressed as mean ± SD. Proportion of the cells in G0/G1,

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S, and G2/M phases were analyzed by the Flowjo software.

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Cell apoptotic analysis by flow cytometry

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Cellular apoptosis was determined following the manufacturer’s instruction of

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Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) and finally analyzed

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by FACScalibur Flow Cytometer (Becton Dickinson).19 Cells were incubated in the

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medium containing 10-G (30 µM) or 5-FU (50 µM) for 12, 24 and 48 h, respectively. 10


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Then, the cells were harvested by trypsinization (0.25% trypsin without EDTA)

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(Invitrogen), and washed twice with cold PBS. The solution containing 5 µL of

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annexin V-FITC and 10 µL of propidium iodide was added to the cells, re-suspended

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in 400 µL binding buffer, and incubated in the dark at 4°C for 10 min. Finally, the

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cells were detected with flow cytometer within 1 h. All assays were performed at least

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three times.

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Assay for RT-qPCR

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Total RNA extracted using Trizol Reagent (Invitrogen, Life Technologies, USA)

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followed by first-strand cDNA synthesis using the Prime Script 1st Strand cDNA

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Synthesis kit and the Oligo dT-adaptor primer in a series of standard 10 µL reverse

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transcription reactions. Changes in the steady-state expression concentration of

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mRNA in cyclin A, cyclin B1, cyclin D1, cyclin E1, CDK-1, CDK-2, CDK-4, CDK-6,

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p15, p16, p21, p27, GSK-3β, and β-catenin were evaluated by reverse-transcription

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PCR (RT-qPCR), which was carried out using EvaGreen Master Mix (Biotium,

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Hayward, CA, USA).32 The primers are presented in Table 1. RT-qPCR was

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performed using the ABI Step One Plus system (Applied Biosystems) followed by

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melting curve analysis with the following cycling program: initial activation at 95°C

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for 3 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C

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for 20 s. GAPDH served as a control for sample loading and integrity.

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Assay for Western blotting

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The method of Western blot was according to Lin et al.32 with some modification.

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Following the treatment with 10-G (15 µM, 30 µM and 50 µM) for 48 h, Hela cells 11



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were washed 3 times in cold PBS and lysed with an ice-cold

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radioimmunoprecipitation (RIPA) buffer containing a protein phosphatase inhibitor

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and a complete protease inhibitor mixture for 30 min over ice. To obtain the cytosol

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fraction, the cell lysates were centrifuged at 15,000 rpm/min for 15 min at 4°C. The

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cytosolic proteins were boiled in a loading buffer, and then the denatured proteins

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were separated by sodium dodecyl sulfate–polyacrylamide (SDSP) gel electrophoresis

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and transferred to a 0.45 µm polyvinylidene difluoride (PVDF) membrane. After 2 h

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at room temperature of incubation in a blocking buffer (150 mM NaCl, 20 mM

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Tris-HCl, 0.1% Tween 20, and 5% skim milk, the membranes were incubated with the

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specific primary antibodies overnight at 4°C. Subsequently, the blot was washed 3

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times with TBST (150 mM NaCl, 20 mM Tris-HCl, and 0.1% Tween 20), followed by

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incubated with the appropriate secondary antibody for 3 h.33 Immunoreactivity was

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detected using the Amersham ECL Prime Western Blot detection reagent (GE

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Healthcare, Fairfield, CT, USA) according to the manufacturer’s instructions.

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Statistical analysis

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Statistical analysis was carried out by using SPSS 18.0 software. All the data were

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expressed as mean ± standard deviation (SD) (n ≥ 3). One-way analysis of variance

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(ANOVA) was per-formed using the Origin Lab (Origin Pro 8.0) software at

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significance level p < 0.05.

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Results and discussion

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10-Gingerol separated from Tongling White Ginger 12


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The α-amylase enzyme (0.5%) was used to pre-treat the ginger before extracting the

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crude extraction of ginger; and then, 10-G was separated using HSCCC

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chromatogram with mobile phase of n-hexane-chloroform-methanol-water system.

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The separated 10-G was determined by HPLC (Figure 1A), and the main peak located

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at 24.092 min, which was calculated as 90.06% of the full components. The separated

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sample was further determined with UPLC-TOF-MS/MS analysis, and the quasi

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molecular ions of sample was (MW 350) m/z = 349 [M-H]+ (Figure 1B).

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Identification and recognition of the structure of purified ginger extraction was

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performed with 1H and 13C NMR. The NMR data of the product were obtained as

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followed. 1H NMR (400 MHz) δ: 0.87 (3H, t, J=6.4 Hz, H-14), 1.35-1.66 (14H, m,

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H-7 ~H-14), 1.49 (2H, m, H-6), 2.42-2.65 (2H, m, H-4), 2.75 (2H, dd, J=10 Hz and

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7.8 Hz, H-2), 2.88 (2H, dd, J=20, 7.8 Hz, H-1), 3.91 (3H, s, OCH3), 4.06 (1H, m,

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H-5), 6.69 (1H, dd, J=9.6, 2 Hz, H-6’), 6.72 (1H, s, H-2’), 6.85 (1H, d, 8, H-5’); 13C

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NMR (100 MHz) δ: 14.27 (C-14, q), 22.93 (C-13, t), 25.57 (C-7, t), 29.0, 29.1 (C-11,

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t; C-10, t), 29.3 (C-9, t), 29.71 (C-8, t), 32.46 (C-1, t), 36.54 (C-6, t), 45.57 (C-2, t),

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49.47 (C-4, t), 56.00 (OCH3, q), 67.68 (C-5, d), 112.52(C-2’, d), 114.30 (C-5’, d),

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132.76 (C-1’, s), 144.08 (C-4’, s), 146.45 (C-3’), 211.61 (C-3, s). Based on the above

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data, the obtained product could be identified as 10-G.

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Effect of 10-gingerol on the inhibition of cancer cell proliferation

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CCK-8 assay was used to evaluate the effects of 10-G for dose-dependent manner on

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the proliferation of HeLa cells (Figure 2A,B). The cells were highly sensitive to 10-G

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treatment at IC50 = 29.19 µM and IC80 = 50.87 µM in a concentration-dependent

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manner (Figure 3B). The treatments of 5-FU (50 µM) and starvation were considered

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as control and the inhibition of cells proliferation after 48 h of treatment reached to

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73.52% (5-FU) and 32.38% (starvation), respectively. 40 µM of 10-G showed 70.08%

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inhibition rate on HeLa cells, which is almost identical to the effect of 5-FU, while,

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above 40 µM, the inhibitory effects of 10-G on the proliferation of HeLa cells were

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higher than 5-FU (50 µM) (Figure 3A). Being used as normal cell line, HEK293 cells

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were used as control to determine the effects of 10-G treatment, whereas it almost

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showed no inhibition on the proliferation (Figure 3B). The above results clearly

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demonstrated that 10-G displayed specific anti-cancerous properties, but no inhibition

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on normal cells. The anti-cancerous properties of ginger make it possible to serve as a

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broad-spectrum cytotoxic agent against cancer cell lines, such as prostate LNCaP

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cells,34 breast MDA-MB-231 cells,35 as well as lung A549, ovarian SK-OV-3, and

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melanoma SK-MEL-2 cells.36 As compared to other ginger extracts, 10-G showed the

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remarkable anti-cancerous effects. In our previous study, the IC50 of 10-G (29.19 µM),

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6-G (96.32 µM), 8-G (43.17 µM), crude extract of ginger leaves (165.91 µg/mL) and

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roots (176.58 µg/mL) demonstrated 10-G as the best inhibitor of Hela cell

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proliferation (data not shown). Above results suggest that Tongling White Ginger is

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suitable to be used as an important ingredient for functional food.

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Previous reports suggested that higher alkyl chain leads to increase in lipophilic

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character (log P) of homologous series of gingerols which further greatly impact the

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inhibition of tumor cell proliferation, by affecting the entry of these substances in the 14


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cells through plasma membrane. Subsequently, the higher log P value corresponds to

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the higher permeation in biological membranes.37, 38 Kim and coworkers concluded

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that IC50 for 10-G (13 µM to 40 µΜ) was more effective than 8-G in inhibiting the

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proliferation of human tumor cells lines.36 Similarly, in another study, Wei and

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coworkers also observed the inhibition of HL-60 proliferation by IC50 for 10-G (56.5

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µM) and 8-G (87.9 µM)”.39 Thus, from our results we can conclude that the

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anti-cancerous properties of ginger can be attributed to 10-G.

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Effect of 10-gingerol on the toxicity to cancer cells

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The release of LDH is usually regarded as an index to assess the integrity of cell

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membranes and also to evaluate the efficacy of cytotoxicity on cancer cells by plant

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derived extractions. As shown in Figure 3C, after exposure to 10-G for 48 h, the LDH

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vitalities were quantitatively assessed for the cytotoxicity on treated cells increased

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gradually in dose dependent manner with the comparison between 5-FU and untreated

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cells. When the concentration increased to 100 µM, 10-G displayed the similar

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cytotoxicity on Hela cells by 5-FU (80 µM) (Figure 3C), and as compared to 5-FU,

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10-G had shown remarkable anti-cancer properties.

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Effect of 10-gingerol on cancer cell morphology

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Hela cells subjected to 10-G (30 µM) for 24, 48 and 60 h exhibited significant

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abnormal forms as compared to untreated and 5-FU-treated cells as negative and

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positive controls (Figure 3). After treatment of 10-G for 48 h, Hela cells displayed

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distinct changes as compared to the negative cells. After 60 h, the damage to HeLa 15



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cells by 10-G (30 µM) was found to be more severe than those treated by 5-FU

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(Figure 3) (increased number of dead cells, apoptotic cells or cell spherical

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suspensions). Cells were reduced in number, appeared round and distorted, lost their

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contact with adjacent cells, and more floating cells, pyknosis, and apoptotic bodies

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were observed. Furthermore, appearance of vacuoles in the cytoplasm of treated cells

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was also noticed (Figure 3).

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Effects of 10-gingerol on cancer cell cycle

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The effects of 30 µM 10-G on HeLa cell cycle progression was examined by flow

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cytometric measurement of cellular DNA content (Figure 4A), and the percentage of

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cells in G0/G1 stage was summarized in Figure 4B. With the increase in time, the

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significant changes appeared in treated cells when compared with the untreated at

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G0/G1 stage.

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The HeLa cells stimulated with 10-G (30 µM) revealed a slight increase at the stage

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of G0/G1: 1.8% after 6 h, and quick rise to 9.8% at 12 h. After cultivated with 10-G

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for 18 h, 67.48% HeLa cells were arrested at G0/G1 stage, and apoptotic cells were

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about two times of the cells present in control group (Figure 4B). After treatment with

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10-G for 24 h, the apoptotic cells increased to 33.62%, which could be associated

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with the block from G0/G1-phase progress into S-phase induced by 10-G. The above

331

results suggested that 10-G can induce HeLa cells cycle arrest in G0/G1-phase. The

332

in-depth studies to know more about the mechanisms of blocking would be of great

333

importance.

334

As 10-G arrested Hela cells in G0/G1 phase, we further studied the molecular 16


Page 16 of 38

Page 17 of 38


335

regulation mechanism by using different concentrations of 10-G, and also by

336

evaluating expression of several important cell cycle-related genes (cyclin A, cyclin

337

B1, cyclin D1, cyclin E1, CDK-1, CDK-2, CDK-4, CDK-6, p15, p16, p21, p27,

338

GSK-3β, and β-catenin) using RT-qPCR analysis. As shown in Figure 4C, cyclin A,

339

cyclin D1, cyclin E1 CDK-2, CDK-4, CDK-6, p15 and p21 mRNA expression was

340

significantly decreased in a dose-dependent manner, while CDK-1, p16, p27, GSK-3β,

341

and β-catenin mRNA expression presented a slight decrease, and nevertheless the

342

cyclin B1 was not evidently changed.

343

Subsequently, the effects of 10-G on the protein levels of the above target genes were

344

studied by western blot analysis. Our results showed the decrease in cyclin A and

345

cyclin D1 protein levels in treated cells (Figure 4D,E), which are the essential markers

346

of G1/S phase, whereas, the cyclin E1 protein had shown on alteration (Figure 4D,E),

347

Similarly, the expression of cyclin B1 as well as the G2/M phase marker did not show

348

any significant change (Figure 4D,E).

349

These results lead to an understanding that 10-G play an important role in controlling

350

the expression of cycle-related targets both at the transcriptional and

351

posttranscriptional level in cancer cells.

352

Effects of 10-gingerol on cancer cell apoptosis

353

It is well-known that apoptosis is implicated for multistage carcinogenesis and

354

phytochemical complexity of plant foods conferring health-promoting benefits

355

including chemo preventive and anticancer effects. To investigate the effects of

356

cytotoxicity of apoptosis by 10-G, the ratios of the sum of apoptotic cells after 17



357

exposure were assessed by flow cytometry using annexin V-FITC and PI labeling

358

after treatments for 12, 24 and 48 h (Figure 5). The apoptosis cells distributed in early

359

stage and late stage were summarized in Table 2. Compared to the negative control,

360

the apoptotic cells induced by 10-G increased dramatically (Figure 5 and Table 2).

361

The apoptotic cells showed no distinctly different attributes after treatment for 12 h

362

with 5-FU and 10-G. When the exposure time was extended, the apoptotic cells

363

numbers (treated by 10-G) were higher to those treated by 5-FU. Particularly, after 48

364

h, the apoptotic cells both in early and late stage were higher than corresponding

365

numbers by 5-FU (Figure 5 and Table 2). The above results indicated that 10-G can

366

induce apoptosis of cancer HeLa cells. Previously confirmed anti-cancerous activities

367

of 10-G against HCT116 human colon cancer cells revealed that inhibition of the

368

proliferation of cells was accompanied by the morphological changes indicative of

369

apoptosis.18

370

Apoptosis is biologically regulated process which comprises two major pathways: the

371

extrinsic and intrinsic pathway.40 Death receptor 3 and 5 (DR3 and DR5), also known

372

as tumor necrosis factor receptors are present on cell surface and mediate apoptotic

373

signaling and differentiation.41 Our study revealed that, 10-G could significantly up

374

regulate both the apoptotic proteins (DR3 and DR5) and resulted in the activation of

375

apoptosis indicators (cleaved caspases-3, -8, and -9) (Figure 6). The increased

376

expression of the caspase cascade leads to 10-G induced apoptosis process.

377

Furthermore, the expression of cytochrome c, Bid, Bad, Bax and Bcl-2 was evaluated

378

to study the mitochondria dependent pathway and significant increase in the 18


Page 18 of 38

Page 19 of 38


379

expression of these proteins was observed (Figure 6) except Bcl-2 which decreased

380

significantly followed by slight decrease in Bid and latter resulted in increased

381

cleaved Bid which ultimately activates Bax. Our results conclude that 10-G activated

382

the intrinsic apoptotic signaling pathways in the Hela cells and led to cell death due to

383

mitochondrial dysfunction.

384

PI3K/AKT pathway as a vital component in the regulation of tumorigenesis and its

385

progression as well as known as upstream signaling molecules of the mTOR pathway

386

get activated on their specific sites through phosphorylation.32 The anti-cancerous

387

activities of 10-G could be associated with a control of signal transduction of

388

PI3K/AKT pathway and this led us to investigate the change of the survival pathway

389

associated proteins. Previous studies suggested that 6-gingerol had no effect on the

390

expression of PI3K, p85α, however, 6-gingerol increased phosphorylation of AKT,

391

which is regulated by PI3K17. Activated AKT promotes the cell survival by

392

anti-apoptotic mechanism and also inactivates the proapoptotic proteins.17 Our present

393

study showed treated cells significantly inhibited the phosphorylation of PI3K (Figure

394

6). The decreases in phosphorylation level of AKT and P70S6K down regulated PI3K

395

while triggered the mTOR (Figure 6). On the other hand, PI3K also changed PKCε,

396

thus regulated the decrease of NF-kβ expression (Figure 6). As known previously, that

397

10-G affected Ca2+ and led to cell death in human colorectal cancer cells,4 and our

398

results showed that it led to induction of AMPK (1.8-fold increase) which finally

399

inhibited mTOR phosphorylation (0.6-fold decrease) signaling.

400

The above results confirmed the anti-cancerous potential of 10-G, and suggested that 19



401

Tongling White Ginger can be utilized as functional food for anticancer effects. We

402

also emphasize the exploitation and utilization of fresh ginger or its components to be

403

explored for other health disorders or ailments and also to preserve the native bio

404

heritage and medicinal properties of native Chinese plants by using suitable animal

405

models followed by clinical trials. This study would be an asset to establish synergy

406

of 10-G and other medicinal plant products for future applications.

407

Effect of inhibitors targeting signal transduction pathway on 10-G induced

408

apoptosis

409

To gain insights into the relationship between 10-G and apoptosis-related pathways,

410

we used the p53 inhibitor (Pifithrin-µ), PI3K inhibitor (Wortmannin), AKT inhibitor

411

(Deguelin), AMPK inhibitor (WZ4003) and mTOR inhibitor (Temsirolimus) to

412

pretreat the Hela cells before 10-G treatment. As shown in Figure 7, these results

413

indicated that treatment with these five inhibitors to a certain extent, could influence

414

the expression of their corresponding proteins. Particularly, Pifithrin-µ and WZ4003

415

dramatically decreased 10-G induced activation of the p53 and p-AMPK protein,

416

respectively. Moreover, cells pre-treatment with Wortmannin could reduce the

417

expression level of p-PI3K, followed by slight augmentation in the inhibitory effects

418

of 10-G on Hela cells. In addition, exposure with Deguelin and Temsirolimus could

419

weakened the inhibitory attributes of 10-G on the expression of p-AKT and p-mTOR.

420

Thus, we speculated, it might be due to their special structure-activity relationship

421

between 10-G and inhibitor, which caused the corresponding competitive binding and

422

masked their synergetic effect. On the other hand, compared with 10-G treatment 20


Page 20 of 38

Page 21 of 38


423

alone, 10-G significantly enhanced the inhibition effect of Deguelin and Temsirolimus

424

on correspondent protein expression. Our results further confirmed 10-G induced

425

apoptosis by PI3K/AKT/AMPK/mTOR signaling pathways in Hela cells.

426

Overall results showed that 10-G triggered mTOR mediated cell apoptosis by

427

inhibiting PI3K/AKT and activating AMPK. After analyzing the cell cycle and

428

apoptosis mechanisms of 10-G, present investigation presented an outline of

429

mechanisms involved in anti-cancerous properties of 10-G (Figure 8).

430

Abbreviations Used 10-G

10-Gingerol

HPLC

High Performance Liquid Chromatography

HSCCC

High-speed Countercurrent Chromatography

UPLC-TOF-MS/MS

Ultra-Performance Liquid Chromatography Time-of-Flight Mass Spectrometer

NMR

Nuclear Magnetic Resonance

DMEM

Dulbecco’s Minimum Essential Medium

FBS

Fetal bovine serum

5-FU

Fluorouracil

CCK-8

Cell Counting kit-8

LDH

Lactate dehydrogenase

PI

Propidium iodide

431

Acknowledgements

432

This study was supported by the Major Projects of Science and Technology in Anhui

433

Province (15czz03115), the grants from the National Natural Science Foundation of 21



434

China (31371947 and 31272111), the Key projects of Natural Science Research of

435

Anhui Province (KJ2016A575) and the Special Fund for Agro-scientific Research in

436

the Public Interest of China (201403064).

437

There is no conflict of interest to declare.

438

References

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471

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GSK-3beta and beta-catenin pathway in A549 cell line. Chem Biol Interact 2016, 258, 257-65. (16) Ho, S. C.; Chang, K. S.; Lin, C. C. Anti-neuroinflammatory capacity of fresh ginger is attributed mainly to 10-gingerol. Food Chem 2013, 141, 3183-91. (17) Park, Y. J.; Wen, J.; Bang, S.; Park, S. W.; Song, S. Y. 6-Gingerol induces cell cycle arrest and cell death of mutant p53-expressing pancreatic cancer cells. Yonsei Med J 2006, 47, 688-97. (18) Ryu, M. J.; Chung, H. S. [10]-Gingerol induces mitochondrial apoptosis through activation of MAPK pathway in HCT116 human colon cancer cells. In Vitro Cell Dev Biol Anim 2015, 51, 92-101. (19) Feng, Y.; Xu, X.; Zhang, Y.; Ding, J.; Wang, Y.; Zhang, X.; Wu, Z.; Kang, L.; Liang, Y.; Zhou, L.; Song, S.; Zhao, K.; Ye, Q. HPIP is upregulated in colorectal cancer and regulates colorectal cancer cell proliferation, apoptosis and invasion. Sci Rep 2015, 5, 9429. (20) Thornberry, N. A.; Rano, T. A.; Peterson, E. P.; Rasper, D. M.; Timkey, T.; Garcia-Calvo, M.; Houtzager, V. M.; Nordstrom, P. A.; Roy, S.; Vaillancourt, J. P.; Chapman, K. T.; Nicholson, D. W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem 1997, 272, 17907-11. (21) Balaban, R. S.; Nemoto, S.; Finkel, T. Mitochondria, oxidants, and aging. Cell 2005, 120, 483-95. (22) Reuter, S.; Eifes, S.; Dicato, M.; Aggarwal, B. B.; Diederich, M. Modulation of anti-apoptotic and survival pathways by curcumin as a strategy to induce apoptosis in cancer cells. Biochem Pharmacol 2008, 76, 1340-51. (23) Johnson, J. J.; Mukhtar, H. Curcumin for chemoprevention of colon cancer. Cancer Lett 2007, 255, 170-81. (24) Nagendra chari, K. L.; Manasa, D.; Srinivas, P.; Sowbhagya, H. B. Enzyme-assisted extraction of bioactive compounds from ginger (Zingiber officinale Roscoe). Food Chem 2013, 139, 509-514. (25) Zhan, K.; Xu, K.; Yin, H. Preparative separation and purification of gingerols from ginger (Zingiber officinale Roscoe) by high-speed counter-current chromatography. Food Chem 2011, 126, 1959-1963. (26) Pawar, N.; Pai, S.; Nimbalkar, M.; Dixit, G. RP-HPLC analysis of phenolic antioxidant compound 6-gingerol from different ginger cultivars. Food Chem 2011, 126, 1330-1336. (27) Lv, L.; Chen, H.; Soroka, D.; Chen, X.; Leung, T.; Sang, S. 6-gingerdiols as the major metabolites of 6-gingerol in cancer cells and in mice and their cytotoxic effects on human cancer cells. J Agric Food Chem 2012, 60, 11372-7. (28) Jung, J. H.; Lee, J. O.; Kim, J. H.; Lee, S. K.; You, G. Y.; Park, S. H.; Park, J. M.; Kim, E. K.; Suh, P. G.; An, J. K.; Kim, H. S. Quercetin suppresses HeLa cell viability via AMPK-induced HSP70 and EGFR down-regulation. J Cell Physiol 2010, 223, 408-14. (29) Johnson, N.; Li, Y. C.; Walton, Z. E.; Cheng, K. A.; Li, D.; Rodig, S. J.; Moreau, L. A.; Unitt, C.; Bronson, R. T.; Thomas, H. D.; Newell, D. R.; D'Andrea, A. D.; Curtin, N. J.; Wong, K. K.; Shapiro, G. I. Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition. Nat Med 2011, 17, 875-82. (30) Wakatsuki, S.; Furuno, A.; Ohshima, M.; Araki, T. Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration. J Cell Biol 2015, 211, 881-96. (31) Petrocca, F.; Visone, R.; Onelli, M. R.; Shah, M. H.; Nicoloso, M. S.; de Martino, I.; Iliopoulos, D.; Pilozzi, E.; Liu, C. G.; Negrini, M.; Cavazzini, L.; Volinia, S.; Alder, H.; Ruco, L. P.; Baldassarre, G.; Croce, C. M.; Vecchione, A. E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 2008, 13, 272-86. (32) Lin, H.-C.; Lin, M.-H.; Liao, J.-H.; Wu, T.-H.; Lee, T.-H.; Mi, F.-L.; Wu, C.-H.; Chen, K.-C.; Cheng, C.-H.; Lin, C.-W. Antroquinonol, a Ubiquinone Derivative from the Mushroom Antrodia camphorata, 23



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543

24


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544

Figure captions

545

Figure 1 HPLC chromatograms and Mass spectrum of extracts from Tongling White

546

Ginger. (A) HPLC chromatograms; (B) Mass spectrum. Column:

547

Alltech-C18 column with 5 µm particle size; mobile phase: acetonitrile:

548

water (58:42); flow rate: 0.8 mL/min; injected volume: 20 µL samples

549

solution; detection wavelength: 280nm.

550

Figure 2 The inhibition and cytotoxicity assessment of 10-gingerol on Hela cells. (A)

551

inhibition of 10-gingerol on the growth of HeLa cells; (B) inhibition curve

552

fitting, HeLa and HEK293 cells were used to analysis; (C) the cytotoxicity of

553

10-gingerol and 5-FU on HeLa cells. All the data were collected after

554

treatment for 48 h, and expressed as mean ± SD of three replicates.

555

Figure 3 The morphological changes of HeLa cell on exposure to 10-gingerol. These

556

were observed by an Inverted Optic Microscope (original magnification ×40).

557

Cells were reduced in number, appeared round and distorted, lost their

558

contact with adjacent cells, and more floating cells (green arrow), pyknosis

559

(white arrow), and apoptotic bodies (blue arrow) were observed. Furthermore,

560

appearance of vacuoles in the cytoplasm of treated cells (yellow arrow) was

561

also noticed. The concentration of 10-gingerol and 5-FU used for treatment

562

of cells were 30 µM and 50 µM, respectively.

563

Figure 4 (A) 10-Gingerol induces cell cycle arrest in HeLa cell lines. The

564

concentration of 10-gingerol used for treatment of cells was 30 µM. (B) The

565

rate of cell cycle arrest in apoptosis and G0/G1-phase. (C) The mRNA

566

expression of cell cycle related genes with increasing concentrations of 25



567

10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated

568

cells. Relative gene-expression levels are expressed with GAPDH as an

569

internal reference. (D) Effect of 10-gingerol on phosphorylation of cell cycle

570

related proteins in Hela cells treated for 48 h analyzed by western blot using

571

tubulin as an internal control. (E) The expression level of the targeted

572

proteins with increasing concentrations of 10-gingerol (15 µM, 30 µM and

573

50 µM) for 48 h as compared to untreated cells. Each value is presented as a

574

mean ± standard deviation (n = 3). Values of a-d represent significantly

575

different under different treatments within same gene, P < 0.05.

576

Figure 5 The effects of 10-gingerol on HeLa cell apoptosis. The concentration of

577

10-gingerol and 5-FU used for treatment of cells were 30 µM and 50 µM,

578

respectively.

579

Figure 6 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in

580

Hela cells treated for 48 h analyzed by western blot using tubulin as an

581

internal control. (B) The expression level of the targeted proteins with

582

increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h

583

as compared to untreated cells. Each value is presented as a mean ± standard

584

deviation (n = 3). Values of a-d represent significantly different under

585

different treatments within same gene, P < 0.05.

586

Figure 7 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in

587

Hela cells treated with or without inhibitors (Pifithrin-µ for p53, Wortmannin

588

for PI3K, Deguelin for AKT, WZ4003 for AMPK and Temsirolimus for 26


Page 26 of 38

Page 27 of 38


589

mTOR) by western blot using tubulin as an internal control. (B) The

590

expression level of the targeted proteins treated by 10-gingerol (with or

591

without inhibitor) at 30 µM as compared to untreated cells. Each value is

592

presented as a mean ± standard deviation (n = 3). Values of a-d represent

593

significantly different under different treatments within same gene, P < 0.05

594 595

Figure 8 Proposed mechanism for the effects of 10-gingerol on cell cycle and apoptosis with a series of pathways involved.

596

27



597 598

Table 1 Primers for real-time PCR Gene Cyclin A Cyclin B1 Cyclin D1 Cyclin E1 CDK-1 p21 p27 GAPDH CDK-2 CDK-4 CDK-6 P15 P16 GSK-3β β-catenin

Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

Sequence (5’-3’) AGACTGAGTGGTTGGATGGCA TGTCCACAGTCAGCAATGGTG AAAGGCGTAACTCGAATGGA CCGACCTTTTATTGAAGAGCA ATGGAACACCAGCTCCTGTGCTGC TCAGATGTCCACGTCCCGCACGT GGATTATTGCACCATCCAGAGGCT CTTGTGTCGCCATATACCGGTCAA TCCGCAACAGGGAAGAAC GAGCCTTTTTAGATGGCTGCT CCACAGCGATATCCAGACATTC GAAGTCAAAGTTCCACCGTTCTC AGCGACCTGCTGCAGAAGAT TTACGTCTGGCGTCGAAGGC TGCCCTCAACGACCACTTTG TACTCCTTGGAGGCCATGTG CTTTGGAGTCCCTGTCCGTA CGAAAGATCCGGAAGAGTTG TGCACAGTGTCACGAACAGA ACCTCGGAGAAGCTGAAACA CATCGTTCACCGAGATCTGA CCAACACTCCACATGTCCAC GCGGCAGCTCCTGGAAG GGGTCGGCACAGTTGG CTTCCTGGACACGCTGGT ATCTATGCGGGCATGGTTACT TCCATTCCTTTGGGATCTGCC ATCAGCTCTGGTGCCCTGTAGTAC GCTGATTTGATGGAGTTGGACATGG GCCAAACGCTGGACATTAGTGG

599

28


Page 28 of 38

Page 29 of 38


600 601

Table 2 The Statistical analysis of effects of 10-gingerol on HeLa cell apoptosis

602 12 h of treatment Rate of cells

24 h of treatment

Early Late stage Normal Normal stage apoptosis cells cells (%)apoptosis cells (%) (%) cells (%)

48 h of treatment

Early Late stage Normal stage apoptosis cells apoptosis cells (%) (%) cells (%)

Early Late stage stage apoptosis apoptosis cells (%) cells (%)

Control

93.76

1.10

2.04

87.11

2.36

6.58

82.36

2.68

10.40

5-FU

86.07

1.84

8.06

72.27

4.38

10.05

53.91

7.26

16.51

10-gingerol 72.98

5.19

8.78

63.19

4.33

22.31

33.82

16.09

26.35

603

29



Figure 1 HPLC chromatograms and Mass spectrum of extracts from Tongling White Ginger. (A) HPLC chromatograms; (B) Mass spectrum. Column: Alltech-C18 column with 5 µm particle size; mobile phase: acetonitrile: water (58:42); flow rate: 0.8 mL/min; injected volume: 20 µL samples solution; detection wavelength: 280nm. 177x199mm (300 x 300 DPI)


Page 30 of 38

Page 31 of 38


Figure 2 The inhibition and cytotoxicity assessment of 10-gingerol on Hela cells. (A) inhibition of 10-gingerol on the growth of HeLa cells; (B) inhibition curve fitting, HeLa and HEK293 cells were used to analysis; (C) the cytotoxicity of 10-gingerol and 5-FU on HeLa cells. All the data were collected after treatment for 48 h, and expressed as mean ± SD of three replicates. 160x47mm (300 x 300 DPI)



Figure 3 The morphological changes of HeLa cell on exposure to 10-gingerol. These were observed by an Inverted Optic Microscope (original magnification ×40). Cells were reduced in number, appeared round and distorted, lost their contact with adjacent cells, and more floating cells (green arrow), pyknosis (white arrow), and apoptotic bodies (blue arrow) were observed. Furthermore, appearance of vacuoles in the cytoplasm of treated cells (yellow arrow) was also noticed. The concentration of 10-gingerol and 5-FU used for treatment of cells were 30 µM and 50 µM, respectively. 277x190mm (300 x 300 DPI)


Page 32 of 38

Page 33 of 38


Figure 4 (A) 10-Gingerol induces cell cycle arrest in HeLa cell lines. The concentration of 10-gingerol used for treatment of cells was 30 µM. (B) The rate of cell cycle arrest in apoptosis and G0/G1-phase. (C) The mRNA expression of cell cycle related genes with increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated cells. Relative gene-expression levels are expressed with GAPDH as an internal reference. (D) Effect of 10-gingerol on phosphorylation of cell cycle related proteins in Hela cells treated for 48 h analyzed by western blot using tubulin as an internal control. (E) The expression level of the targeted proteins with increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated cells. Each value is presented as a mean ± standard deviation (n = 3). Values of a-d represent significantly different under different treatments within same gene, P < 0.05. 160x187mm (300 x 300 DPI)



Figure 5 The effects of 10-gingerol on HeLa cell apoptosis. The concentration of 10-gingerol and 5-FU used for treatment of cells were 30 µM and 50 µM, respectively. 160x133mm (300 x 300 DPI)


Page 34 of 38

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Figure 6 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in Hela cells treated for 48 h analyzed by western blot using tubulin as an internal control. (B) The expression level of the targeted proteins with increasing concentrations of 10-gingerol (15 µM, 30 µM and 50 µM) for 48 h as compared to untreated cells. Each value is presented as a mean ± standard deviation (n = 3). Values of a-d represent significantly different under different treatments within same gene, P < 0.05. 160x198mm (300 x 300 DPI)



Figure 7 (A) Effect of 10-gingerol on phosphorylation of apoptosis related proteins in Hela cells treated with or without inhibitors (Pifithrin-µ for p53, Wortmannin for PI3K, Deguelin for AKT, WZ4003 for AMPK and Temsirolimus for mTOR) by western blot using tubulin as an internal control. (B) The expression level of the targeted proteins treated by 10-gingerol (with or without inhibitor) at 30 µM as compared to untreated cells. Each value is presented as a mean ± standard deviation (n = 3). Values of a-d represent significantly different under different treatments within same gene, P < 0.05. 160x80mm (300 x 300 DPI)


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Figure 8 Proposed mechanism for the effects of 10-gingerol on cell cycle and apoptosis with a series of pathways involved. 160x137mm (300 x 300 DPI)



Graphic for table of contents 45x24mm (300 x 300 DPI)


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Tongling White Ginger - ACS Publications - American Chemical

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