Chitosan Oligosaccharides Induce Apoptosis in Human Renal

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Bioactive Constituents, Metabolites, and Functions

Chitosan oligosaccharides induce apoptosis in human renal carcinoma via ROS-dependent ER stress Xingchen Zhai, Shoujun Yuan, Xin Yang, Pan Zou, Linna Li, Guoyou Li, Yong Shao, A. M. Abd El-Aty, Ahmet Hac#müftüo#lu, and Jing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06941 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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Journal of Agricultural and Food Chemistry

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Chitosan

oligosaccharides

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ROS-dependent ER stress

3

Xingchen Zhai

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Yong Shao ‡, A. M. Abd El-Aty ‖,¶, Ahmet Hacımüftüoğlu ¶, Jing Wang **,†, ‡

5

†Department

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Engineering, Harbin Institute of Technology, No. 92 West Dazhi Street, Nangang District,

7

Harbin, Heilongjiang Province 150090, PR China

8

‡Key

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Technology for Agro-Product, Chinese Academy of Agricultural Sciences, No. 12

†,‡,§,

induce

apoptosis

in

human

Shoujun Yuan §, Xin Yang *,† , Pan Zou

†,‡,

renal

carcinoma

via

Linna Li §, Guoyou Li §,

of Food Sciences and Engineering, School of Chemistry and Chemical

Laboratory of Agro-Product Quality and Safety, Institute of Quality Standard & Testing

10

Zhongguancun South Street, Haidian District, Beijing 100081, PR China

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§Department

12

100081 Beijing, PR China

13

‖

14

12211-Giza, Egypt

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¶Department

16

25240-Erzurum, Turkey

of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine,

Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University,

of

Medical

Pharmacology,

Medical

Faculty,

Ataturk

University,

17 18

1

ACS Paragon Plus Environment


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Abstract

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In recent years, various studies have confirmed the role of natural products as effective

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cancer prevention and treatment drug. The present study demonstrated chitosan

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oligosaccharide (COS) from shells of shrimp and crab, caused an inhibitory effect on the

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proliferation of human renal carcinoma in vitro and in vivo. First, the in vivo biodistribution

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of COS was investigated by the synthesis of cyanine 7-labelled COS (COS-Cy7) following

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tail vein injection. The kidney was found to be major target organ. Then, the impacts on renal

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carcinoma cell proliferation, apoptosis and ROS production were observed in vitro, and an

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orthotopic xenograft tumour model was designed to evaluate the antitumour efficacy of COS

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in vivo. In renal carcinoma cells, COS induced G2/M phase arrest and apoptosis in a

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ROS-dependent fashion. COS significantly promoted mRNA expression of Nrf2 and Nrf2

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target genes, such as HMOX1, GCLM, and SLC7A11. Additionally, COS significantly

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upregulated the protein expression of GRP78, PERK, eIF2α, ATF4, CHOP and Cyt c, which

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justified the activation of the ER stress signalling pathway. In vivo, COS repressed tumour

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growth and induced apoptosis and ROS accumulation, consistent with the in vitro results.

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Taken together, COS repressed human renal carcinoma growth and induced apoptosis both in

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vitro and in vivo, mainly via ROS-dependent ER stress pathways.

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Keywords: Chitosan oligosaccharides; Renal carcinoma orthotopic xenograft; Antitumor

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effect; ER stress; ROS accumulation

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Introduction

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The regulation of oxidative stress is a crucial factor in both tumour development and

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responses to anticancer therapy. Oxidative stress can be defined as a force that imposes a

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biochemical or biophysical perturbation that requires homeostatic correction to preserve

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physiological balance 1. Reactive oxygen species (ROS) is such a response to perturbations.

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To date, the biological roles of ROS in the body remain ambiguous 2. However, due to

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metabolic and signalling aberrations, cancer cells usually have higher levels of ROS

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compared to normal cells. This aspect offers an interesting therapeutic window, because

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cancer cells might be more sensitive than normal cells to agents that cause further

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accumulation of ROS 3. Agents can have tumour-suppressive effects through oxidative stress.

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ROS are mainly generated in mitochondria, but an increasing number of studies suggest that

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endoplasmic reticulum (ER) stress is also a significant source of ROS. The ER is the largest

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membranous organelle of eukaryotic cells, and its main function is to fold and process

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nascent proteins that enter into it. ER function is disrupted when cells are stimulated by

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different pathological or physiological stimuli, such as oxidative stress, DNA damage or

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hypoxia 4.

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Currently, there is a trend in searching for naturally occurring anticancer agents because

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they are considered biologically friendly. So far, approximately half of the approved

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anticancer agents were partially and/or entirely originated from plant-derived natural products

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5.

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agents and/or adjuvant treatments can improve the therapeutic efficacy of commercially

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available chemotherapeutic agents and reduce the side effects 6. Chitosan oligosaccharide

The development of efficacious, targeted therapies from natural sources as new antitumour

3



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(COS) has been approved as a new food ingredient according to the relevant provisions of the

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“Food Safety Law of China” in 2014. COS and its derivatives possess several biological

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activities7, 8. COS not only manifests favourable antitumour activities but also exerts potential

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as an adjuvant therapy with other chemotherapeutic agents, such as sitagliptin

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cyclophosphamide 10.

9

and

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During research, most of the animal models used were a subcutaneously implanted

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tumour model, which is an ideal model for assessing the antitumour efficacy of experimental

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agents, offering the advantage of easy quantification of tumour volume. However,

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subcutaneously implanted tumours fail to reflect the actual characteristics of clinical tumours.

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The orthotopic implantation model is the most closely related model to clinical tumour

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pathophysiological aspects with practical applications. Hoffman

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implantation models offer a link between pre-clinical studies and clinical trials/drug

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development. Orthotopic implantation models are superior to subcutaneous implantation

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models, especially in tumour metastasis and drug sensitivity studies. Therefore, based on the

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biodistribution of COS, an orthotopic xenograft model of renal carcinoma with fluorescent

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labeling cells, was established to detect the pharmacological effects. Based on the above

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findings, we show the link between the ER stress response and cell death through important

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cellular signalling pathways by qRT-PCR and western blotting, to indicate that COS can

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induce apoptosis via ROS-dependent ER stress pathways in vitro and in vivo.

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found that orthotopic

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Materials and Methods

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Drugs, chemicals, and reagents

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RPMI-1640 medium and PBS (pH 7.4 basic) as well as fetal bovine serum (FBS) were

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bought from Gibco (Grand Island, NY, USA). Penicillin and streptomycin were obtained

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from Thermo Fisher Scientific (Waltham, MA, USA). Cyanine 7-NHS (Cy7-NHS, a

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near-infrared dye) was supplied by Xi’an ruixi Biological Technology Company (Xi’an,

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China). N-acetyl cysteine (NAC) was acquired from Aladdin Biochemical Technology

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Company (Shanghai, China). The COS used in this study was degraded from commercial

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chitosan (medical grade chitosan, Zhejiang Fengrun Biotech Co., Taizhou, Zhejiang, China.

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Table S1) and had an average molecular weight of 1000 Da. The degree of polymerization

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was 2-6, and the degree of deacetylation was 95.3±1.3%

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solvents used throughout the experimental work were of analytical reagent grade and secured

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from Sinopharm Chemical Reagent Company (Beijing, China).

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Cell culture and viability assay

12, 13.

All other chemicals and

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A human renal carcinoma cell line (KCC853), generously offered by the Department of

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Pharmacology and Toxicology, Beijing Institute of Radiation Medicine (Beijing, China), was

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cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100

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μg/mL streptomycin. Cells were maintained at 37°C in a humidified incubator with a 5% CO2

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atmosphere.

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KCC853 cells were seeded in 96-well plates and stimulated with various concentrations 5



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of COS (0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/mL) for 72 h (100 μL of 10 μM

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NAC for 2 h before COS, in the case of NAC pre-treatment). MTT assays were used to

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evaluate the inhibition rates. One hundred microlitres of fresh medium containing 0.5 mg/mL

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MTT was added per well and incubated at 37°C for 4 h under 5% CO2. The absorbance was

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measured using an automatic microplate reader (BMG LABTECH, Ortenberg, Germany) at

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570 nm after formazan crystals were solubilized in DMSO. Cell viability is expressed as a

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percentage of the control, and the dose-response function was analysed using Origin 8.5

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software.

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Flow cytometry analysis of apoptosis and cell cycle distribution

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KCC853 cells were seeded in six-well plates and allowed to adhere for 24 h. Afterward,

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different concentrations of COS (0, 250, 500, or 1000 μg/mL) were added for an additional

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24 h (100 μL of 10 μM NAC for 2 h before COS, in NAC pre-treatment). Non-adherent and

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adherent cells were harvested together. Cells were washed with cold PBS and then suspended

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in 100 μL of Annexin V binding buffer containing 1 μL of FITC Annexin V and 2 μL of

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propidium iodide (PI) dyes (Biolegend, San Diego, CA, USA). After incubation for 15 min in

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the dark at room temperature, the cells were washed and re-suspended in 400 μL of binding

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buffer for flow cytometry analysis (Millipore, Burlington, MA, USA).

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To analyse the effects of COS on cell cycle distribution, KCC853 cells were treated as

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mentioned above, collected, washed and suspended in PBS, and then fixed with 50% ethanol

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for 40 min at 4°C. Subsequently, cells were washed and re-suspended in PBS containing 0.1

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mg/mL RNase at 37°C for 30 min. After centrifugation, cells were finally stained with 50 6


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μg/mL PI in PBS for 5 min before flow cytometry analysis 14. The results were quantified by

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ModFit LT (Verity Software House, USA).

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Comet assay

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KCC853 cells were seeded in six-well plates and allowed to adhere for 24 h prior to

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exposure to 1000 μg/mL COS for an additional 24 h. Thereafter, the cells were collected,

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washed, and processed according to the manufacturer’s protocols.

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Reactive oxygen species (ROS) detection

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Intracellular ROS were measured using H2DCF-DA, the most widely used indicator for

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ROS in cells. The procedures were carried out using a ROS detection Assay Kit (BioVision,

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San Francisco, CA, USA) according to the manufacturer’s guidelines.

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qRT-PCR

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Cells were treated with different concentrations of COS (0, 250, 500 or 1000 μg/mL) for

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48 h, and total RNA was extracted from the control and drug-treated groups using TRizol

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reagent. cDNA was synthesized with a HiFiScript cDNA Synthesis Kit (CWBIO, Taizhou,

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Jiangsu, China) according to the manufacturer’s protocol. qRT-PCR was performed using

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UltraSYBR Mixture (CWBIO, Taizhou, Jiangsu, China). The primers used for the qRT-PCR

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analysis are listed in Table S2.

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Mitochondrial membrane potential (MMP) estimation

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MMP was measured using JC-1 dye, the most widely used molecular probe for

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MMP in cells. The procedures were carried out using a JC-1 detection Assay Kit (Beyotime

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Biotechnology, Shanghai, China) according to the manufacturer’s guidelines. Flow cytometry 7



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was used to quantify the MMP with green fluorescence (JC-1 monomers) and red

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fluorescence (JC-1 aggregates).

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Intracellular calcium level measurement

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Fluo-4AM dye (Beyotime Biotechnology, Shanghai, China) was used to measure

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intracellular calcium levels. Briefly, KCC853 cells were seeded into six-well plates and

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exposed to different concentrations of COS for 48 h. Then, the cells were collected and

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washed with PBS three times. After that, the cells were incubated with PBS containing 1 μM

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working solution of Fluo-4AM at 37°C under darkness for 40 min. Then, the cells were

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collected and resuspended in PBS before flow cytometry measurement.

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Western blotting

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Total protein from KCC853 cells was extracted using RIPA cell lysis buffer. After

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incubation on ice for 20 min, the lysates were centrifuged at 12000 rpm for 15 min at 4°C,

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and the supernatants were collected for Western blot analysis. Protein (40-60 μg) from each

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sample was loaded on mini-protean precast gels (Bio-Rad Laboratories, Hercules, CA, USA)

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and then transferred onto PVDF membranes. After blocking in 5% non-fat milk, the

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membranes were incubated with the primary antibodies overnight at 4°C, followed by the

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secondary antibody. The relative density of each band was analysed by ChemiCapture (Clinx

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Science Instrument, Shanghai, China), and β-actin was used as a loading control to normalize

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protein expression.

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Animal study

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Forty-five nu/nu, specific pathogen-free female mice (weighing 18-22 g) were purchased 8


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from Vital River Laboratory Technology Co. (Beijing, China). Animals were housed under

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normal laboratory conditions (room temperature, 12 h light-dark cycle) with free access to

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food and water ad libitum. All experimental procedures were in line with the Beijing Medical

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Experimental Animal Care Commission. This work was approved by The Laboratory Animal

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Ethics Committee of Beijing Institute of Radiation Medicine (Beijing, China; certificate no.,

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BIRMSPF-120125A).

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Biodistribution of COS

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The biodistribution of COS was performed as previously described with minor

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modifications 15. Cy7-labelled COS (COS-Cy7) was injected through the tail vein into three

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normal nu/nu mice and then imaged under anaesthesia (with isoflurane) at 0.5, 5, 24, 48, and

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72 h post-injection using an IVIS (in vivo imaging system) Spectrum CT instrument

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(PerkinElmer, USA). Three mice were sacrificed at 5, 48, and 72 h post-injection. The heart,

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liver, spleen, lung, kidney, and bladder were immediately dissected and washed with PBS,

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and fluorescence images were acquired. As a control, the same fluorescence intensity of free

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Cy7-NHS (free Cy7) was also intravenously injected through the tail vein into three other

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normal nu/nu mice and imaged at the same time intervals. The in vivo and ex vivo

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fluorescence was detected at excitation and emission wavelengths of 745 nm and 800 nm,

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respectively.

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Xenograft tumour model of human renal carcinoma and treatments

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KCC853-luc-GFP cells, which are KCC853 cells stably transfected with green

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fluorescent protein (GFP) and luciferase (luc) reporter genes, were suspended in 200 μL of 9



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PBS at a density of 2.5×107/mL and then injected subcutaneously into the backs of five nude

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mice. When the volume of the subcutaneous xenograft tumours reached 400-600 mm3,

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tumour tissues were harvested from the hosts and cut into small sections measuring ~2×2×2

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mm3 in size under sterile conditions. The tumour sections were then implanted into the

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subcapsular area of the right kidney of the nude mice as previously described 16. At 10 days

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post-implantation of the tumours, the nude mice were imaged using IVIS Spectrum CT

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following intraperitoneal injection of 150 mg/kg D-luciferin. The animals were allocated into

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four groups (eight per group) according to the fluorescence intensity: the control, COS 10

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mg/kg, COS 20 mg/kg, and COS 40 mg/kg groups. COS was administered daily by

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intraperitoneal injection. The first treatment began on the day of allocating the animals to

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treatment groups, which was considered day 1. Tumour growth was assessed by

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bioluminescence total flux (p/s) and was observed by IVIS Spectrum CT on days 13 and 20.

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Following the last observation on day 20, the mice were sacrificed, and the tumours were

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harvested. Four specimens were fixed in formalin (10%, w/v in phosphate-buffered saline,

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PBS, pH 7.4), and the rest (4 samples) were submerged in liquid nitrogen for cryo-etching. At

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the same time, blood samples were collected, and sera were allowed to clot for 30 min at

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room temperature followed by centrifugation for assessment of biochemical markers,

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including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline

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phosphatase (AKP), blood urea nitrogen (BUN), serum creatinine (Cr), and albumin protein

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(ALB).

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Histological analysis and apoptosis detection with TUNEL in situ 10


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Tumour specimens fixed in liquid nitrogen were cut into two-micrometre thick slices and

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incubated in diluted dihydroethidium (DHE) at 37°C (in the dark) for 30 min. Following

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multiple washes with PBS, the slices were incubated in DAPI staining solution (in the dark)

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for 10 min at room temperature. After several washes in PBS, the slices were then incubated

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in an auto-fluorescence quenching agent for 5 min before being analysed under a

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fluorescence microscope (Nikon Eclipse C1, Tokyo, Japan).

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Hematoxylin and eosin (HE) staining procedures were performed as previously reported

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10.

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-deoxyuridine 5 ′ -triphosphate (dUTP) nick-end labelling (TUNEL) assays to

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detect apoptotic cells. Representative paraffin blocks were cut into two-micrometre thick

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sections, deparaffinized in xylene, rehydrated with a graded series of ethanol, and rinsed in

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PBS. The slices were then incubated in an auto-fluorescence quenching agent for 5 min and

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then washed with water before being incubated in proteinase K for retrieval. In the staining

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process, TUNEL solution was slowly dropped onto the tissue slices and layered with a cover

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to avoid evaporation during incubation. Slices were incubated for 60 min at 37°C in the dark.

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Following multiple washes in PBS, the slices were incubated in DAPI staining solution for 10

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min at room temperature in the dark. Slides were washed in PBS prior to analysis via

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fluorescence microscopy at the proper wavelength (Nikon Eclipse C1, Tokyo, Japan).

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CaseViewer software was used for image analysis.

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Statistical analysis

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Tumour tissues were also used for terminal deoxynucleotidyl transferase-mediated 2 ′

All in vitro experiments were performed in triplicate, and the data are presented as the 11



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mean ± corresponding standard deviations (SDs). One-way analysis of variance (ANOVA)

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followed by Dunnett’s test and two-way ANOVA followed by Bonferroni’s test were used

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to determine significant differences (P

Chitosan Oligosaccharides Induce Apoptosis in Human Renal

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