Phycocyanin reduces proliferation of melanoma cells through

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Phycocyanin reduces proliferation of melanoma cells through downregulating GRB2/ERK signaling Shuai Hao, Shuang Li, Jing Wang, Lei Zhao, Chan Zhang, Weiwei Huang, and Chengtao Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03495 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018

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Journal of Agricultural and Food Chemistry

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Phycocyanin reduces proliferation of melanoma cells through downregulating

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GRB2/ERK signaling

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Shuai Hao †,*, Shuang Li †, Jing Wang †, Lei Zhao †, Chan Zhang †, Weiwei Huang ‡,

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Chengtao Wang †,*

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†

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Engineering and Technology Research Center of Food Additives, Beijing Technology and

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Business University, Beijing, 100048, China

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‡

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Genetron Health (Beijing) Co. Ltd, Beijing, 102208, China

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* Corresponding authors:

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Shuai Hao & Chengtao Wang: Beijing Advanced Innovation Center for Food Nutrition and

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Human Health, Beijing Engineering and Technology Research Center of Food Additives,

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Beijing Technology and Business University, No. 11 Fucheng Road, Haidian District, Beijing,

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100048, China.

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Email address: [email protected] (Shuai Hao);

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[email protected] (Chengtao Wang)

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ACS Paragon Plus Environment


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Abstract

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As a type of functional food additive, phycocyanin is shown to have a potential

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antineoplastic property. However, its underlying anti-cancer mechanism in melanoma cells

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remains unknown. We previously reported a

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proteomic (SiLAD) technology. It could exclusively detect protein synthesis rates via

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labeling of newly expressed proteins by

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analysis of protein variations. In present study, we performed a time course analysis in A375

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melanoma cells after phycocyanin treatment using SiLAD. Protein expression velocities were

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specifically visualized and their regulation modes were dynamically traced. Strikingly, novel

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protein synthesis patterns were discovered at early phase of phycocyanin treatment,

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suggesting possible mechanism of phycocyanin regulation. Furthermore, network analysis

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and phenotype experiments demonstrated that GRB2-ERK1/2 pathway was involved in

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phycocyanin-mediated regulation process and responsible for the proliferation suppression of

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melanoma cell, which could be a therapeutic target for malignant melanoma.

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35

S in vivo/vitro labeling analysis for dynamic pulse

S, providing a high time-resolution method for

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Keywords: phycocyanin, dynamic proteomics, melanoma, protein synthesis rates, ERK

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pathway

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Introduction

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Malignant melanoma, one of the most aggressive forms of skin cancer, has led to a high

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rate of skin cancer-related deaths1. Although lots of efforts have been made to explore the

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therapeutic methods of melanomas, the molecular mechanism underlying development of

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malignant melanoma was still elusive.

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Phycocyanin (PC) is one of the well-known natural functional food additives2, which

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usually derives from the photosynthetic pigment protein in cyanobacteria and Spirulina cells.

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It has been reported that phycocyanin has multiple physiological functions, including

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anti-tumor3, 4, antioxidant5, 6, immunomodulatory7, anti-inflammatory5, 8, anti-bacterial

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activities9 and so on. Particularly, current researches demonstrate that phycocyanin has

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potential

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selenium-containing phycocyanin has antioxidant and anti-proliferative activities in

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melanoma A375 cells10. Baudelet et al. discover that glaucophyte Cyanophora paradoxa

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pigments could efficiently suppress the proliferation of malignant melanoma cells11. It's worth

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mentioning that although the inhibition function of phycocyanin in melanoma cells is well

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known, the mechanism of its network regulation is still unclear. Wu et al. find that ERK and

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p38 MAPK signaling pathways are involved in phycocyanin mediated antimelanogenic effect

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in B16F10 cells12, while they fail to screen the key upstream intermediate regulatory factors.

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Therefore, further investigation on the regulation mechanism of phycocyanin in melanoma is

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essential to develop and improve new therapy for this malignancy.

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The

35

application

in

melanoma

treatment.

Chen

et

al.

have

reported

that

S in vivo/vitro labeling analysis for dynamic proteomic technology (SiLAD) was

established and applied in our former study13, 14. In this method, through pulse labeling on

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35

S-methionine and

35

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cells for 15-30 min by

S-cysteine mixer, the newly expressed proteins

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can be specifically detected and visualized by autoradiography (phosphor imaging, PI). Due

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to the high sensitivity and temporal resolution feature of isotope pulse labeling technique,

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SiLAD could exclusively reveal instantaneous protein variations or protein expression

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velocities during detected biological process14. In this study, to further investigate the precise

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regulation mechanism of phycocyanin in melanoma cells, we traced the protein expressions

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within 48 h in human melanoma A375 cells after phycocyanin treatment by SiLAD for the

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first time. As a result, protein expression velocity was specifically detected, and strikingly,

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novel dynamic regulation rules of phycocyanin were visualized, which provided a distinctive

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perspective for studying the mechanism of phycocyanin and functional foods.

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Materials and Methods

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Cell line and culture condition

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The melanoma A375 cell line was purchased from American Type Culture Collection

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(ATCC, Manassas, VA, USA). A375 cells were cultured in RPMI 1640 medium containing 10%

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FCS, and grown in a humidified incubator with 5% CO2 at 37°C.

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Phycocyanin preparation Phycocyanin was purchased from Envirologix (Portland, ME, USA). The maximum absorption wavelength of dissolved phycocyanin was 618 nm, and its purity was over 95%15.

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siRNA Transfection

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The siRNA Transfection was performed as described in our previous study15. Briefly,

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cells were seeded into a 6-well plate with an appropriate density beforehand, and transfected

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into 80 nM of a siRNA (GenePharma, Shanghai, China) for each well using DhamaFECT 1

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reagent according to the manufacturer’s instructions (Dharmacon, Lafayette, CO, USA).

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Neg. siRNA was used as the negative control.

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The 35S in vitro labeling analysis for dynamic proteomic (SiLAD) technology

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The SiLAD experiment was performed as described in our previous study13-15. Briefly,

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After treated with 6 µM phycocyanin, NEG-722 EasytagTM Express Protein Labeling Mix

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(PerkinElmer, Waltham, MA, USA) was added into medium of A375 cells for 30 min at

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indicated time points (0, 3, 8, 12, 24 and 48 h after phycocyanin treatment). After labeling,

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cells were collected and washed twice (with 250 mM sorvbitol) for protein extraction.

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Proteins were extracted by urea lysis buffer containing 40 mM Tris, 4% CHAPS, 7 M urea, 65

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mM dithiothreitol and 2M thiourea, followed by ultrasonic treatment. The protein supernatant

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solution was obtained after centrifugation for 1 h at 55,000 rpm, and stored at -80°C for

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further use.

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2-DE (two dimensional electrophoresis) was performed to separate the labeled proteins,

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followed by Coomassie Brilliant Blue (CBB) staining and dry gel producing according to our

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previous work13-15. The dry gels were exposed using phosphor imaging plates (Fuji Photo

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Film Co. Japan) in a Pharos FXTM Plus Molecular Imager (Bio-Rad)13. Differential proteins

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were analyzed by ImageMaster 2-D Platinum, followed by matrix-assisted laser

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desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification.

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Western blotting

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Western blotting was performed as described in our former study13-15. Briefly, Cells were

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lysed in RIPA buffer for protein extraction. Equal amounts of protein samples were separated

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by SDS-PAGE, followed by transferred onto polyvinylidene difluoride (PVDF) membranes

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(Millipore, Schwalbach, Germany). Then the primary and secondary antibodies (Cell

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Signaling Technology, Boston, MA, USA) were incubated with PVDF membranes, followed

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by signal detecting using chemiluminescence system (Millipore).

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Immunoprecipitation and phosphor imaging The

protein

expression

velocity

was

verified

by

phosphor

imaging

and

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immunoprecipitation assay according to our previous study15. Briefly, the labeled proteins

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were incubated with G protein coupled glucan beads (Santa Cruz, Dallas, TX, USA) and

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corresponding primary antibodies. After centrifugation, the target proteins binding to the

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beads were collected and subjected to SDS-PAGE. Then the gels were exposed by phosphor

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imaging for signal detection after dried.

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Cell viability assay

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Cell viability was performed as described in our previous study16. Briefly, A375 cells

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were seeded at an appropriate density into 96-well plates the day before phycocyanin addition.

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Then cells were incubated with phycocyanin at indicated final concentrations (0, 2, 4, 6, and 8

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µM) for 24 h. After incubation, MTT was added into each well for 4 h, followed by DMSO

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dissolution. The absorbance was measured at 630 nm and 460 nm. The cell viability was

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shown as the ratio of absorbance reading and its corresponding control cells.

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Proliferation assay

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Cell proliferation assay was performed as described in our former study16. Briefly, after

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incubation with phycocyanin for 24 h, cells were seeded at an appropriate density into 96-well

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plates the day before detection. The A375 cells were incubated with MTT for 4 h, followed by

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SDS-HCl solution addition in each day. The absorbance was detected at 570 nm and 630 nm.

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The proliferation assay lasted for 5 days. Three independent experiments were carried out.

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Statistical analysis

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The statistic difference was analyzed using SPSS software and label by asterisk (*,

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p

Phycocyanin reduces proliferation of melanoma cells through

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