Journal of Drug Targeting

ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20

Brucea javanica oil emulsion alleviates cachexia

induced by Lewis lung cancer cells in mice

Chao Chen & Binbin Wang

To cite this article: Chao Chen & Binbin Wang (2017): Brucea javanica oil emulsion alleviates
cachexia induced by Lewis lung cancer cells in mice, Journal of Drug Targeting, DOI:
10.1080/1061186X.2017.1354003

To link to this article: http://dx.doi.org/10.1080/1061186X.2017.1354003

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Jul 2017.

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 Brucea javanica oil emulsion alleviates cachexia induced by Lewis

lung cancer cells in mice

Chao Chen 1, Binbin Wang 2*

1
Department of Radiotherapy, Zhejiang Provincial Hospital of Traditional Chinese

Medicine, Hangzhou, 310007, Zhejiang Province, China

2
Department of Oncology, Zhejiang Provincial Hospital of Traditional Chinese

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Medicine, Hangzhou, 310007, Zhejiang Province, China

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*Corresponding Author Binbin Wang; Tel & Fax number: +86-571-87071083;

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E-mail: [email protected]; Postal address: Department of Oncology, Zhejiang

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Provincial Hospital of Traditional Chinese Medicine, No.54 on Youdian Road,
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Hangzhou, 310007, Zhejiang Province, China
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 Brucea javanica oil emulsion alleviates cachexia induced by Lewis

lung cancer cells in mice

Abstract. This study was conducted to evaluate the efficacy and possible mechanism of

Brucea javanica oil emulsion (BJOE) on cachexia, by observing changes in related indexes in

mice with cachexia and identifying the genes responsible based on gene chip analysis. In the

BJOE treatment group, body weight loss, tumor growth and metastasis were found obviously

inhibited, food and water intake had markedly increased, and survival time was significantly

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prolonged, as compared to the control group. Moreover, the BJOE witnessed improvement in

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body weight, prevention of tumor metastasis and overall increase in survival time, as

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compared to Indometacin (IND, the positive control medicine). It was also found that TNF- α
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and IL-6 in serum were significantly lower in both groups of BJOE and IND, than in the

control group (P<0.01). Based on the gene expression data, 7 and 6 hub genes of BJOE and
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IND groups were found in the PPI networks, and three common genes comprising of Nmd3,
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Bcl2, and Nhp2l1 were screened. Thus, BJOE could reduce tumor growth and effectively

alleviate cancer cachexia, due to inhibition of pro-inflammatory cytokines. Nmd3, Bcl2,

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Nhp2l1 may be important drug targets, establishing the role of BJOE in the treatment of lung
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cancer induced cachexia.

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Keywords. Brucea javanica oil emulsion, cachexia, drug targets, hub genes, lung cancer,

TNF-α
Introduction

Cancer cachexia is often seen in advanced stages of cancer. It is a wasting syndrome

characterized by a continuous loss of skeletal muscle mass (with or without loss of

fat), resulting in progressive functional impairment [1-3]. It significantly damages the

quality of life, accompanied by weight loss, anorexia, anemia and prolonged

depression [4,5]. It is estimated that 50%-80% of cancer patients experience cachexia,

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and it accounts for up to 20% of all cancer deaths [6]. Commonly used treatments

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include appetite stimulants, anti-depressives, and anti-emetics along with nutritional

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therapy as a part of the best supportive care regimen. However, the side-effects and

intolerance, limit the existing treatment application for cancer cachexia, and research
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on new therapies has become imperative. Traditional Chinese medicines (TCM) are
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compliant, efficient and have lesser-toxicity in cancer patients, and hence have drawn

more attention in clinical practice [7-11].

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Brucea javanica (L.) Merr, belongs to the family Simaroubaceae, and is an

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evergreen shrub widely distributed in Southeast Asia and northern Australia.

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Quassinoids that are extracted from the fruits (or seeds) of B. javanica, as the main
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active ingredients, possess various biological properties, such as antifungal,

anti-oxidative, anti-inflammatory and anticancer properties [12-15]. Recent

investigations have suggested that Brucea javanica could inhibit proliferation or

induce apoptosis in human liver cancer, colon cancer, ovarian cancer, non-solid

tumors such as leukemia, etc. However, its effectiveness in cancer cachexia is still
unknown [16-19].

In this present study the effects of Brucea javanica oil emulsion (BJOE) on the

physiological status of Lewis lung carcinoma cachexia in mice was observed,

including food intake, body weight, tumor growth, lung metastasis, etc., and the

mechanism of BJOE in improving cancer cachexia was explored.

Materials and Methods

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Materials

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BJOE injections, Z19993152, each loaded with 10 ml emulsion, were purchased from
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Zhejiang Jiuxu Pharmaceutical Co., Ltd. Indometacin suppositories (IND),

H11021391, 100 mg each, were purchased from Beijing Twinluck Pharmaceutical Co.,
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Ltd. and were dissolved in normal saline i.e. 0.2 mg/ml. Lewis Lung Carcinoma (LLC)
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cells were introduced from the pharmacology laboratory in China State Institute of

Pharmaceutical Industry (Shanghai). All enzyme-linked immunosorbent assay

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(ELISA) kits were purchased from R&D Systems, Inc. The Ambion Illumina RNA
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Amplification Kit, WG-6 BeadChip, Bead Array Reader and GenomeStudio were
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provided by Illumina, Santiago, California, USA.

Animals

Forty-five male C57 BL/6 mice were provided by Central Lab Animal Inc. (Shanghai,

China) at 6-8 weeks of age (18-22g), and were kept in a pathogen-free environment.
They were randomized into three groups: (A) BJOE group, (B) IND group, (C)

control group (n=15 in each group). Mice in all three groups were injected

subcutaneously with 1.5×106 LLC cells in the anterior axilla on Day 0 and were fed

normally for 24 days. Then BJOE was administered at a dose of 40 ml/kg.bw to the

BJOE group, while the same volume of regular saline solution was administered in

the control group, and IND solution was administered at 5 ml/kg.bw (1 mg/kg.bw).

The concentrations of BJOE and IND were selected as per previous reports [20-23],

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which indicate the doses are optimal for cancer treatment with relatively fewer side

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effects. Moreover, the preliminary experiments with different doses of IND (1

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ml/kg.bw, 5 ml/kg.bw, 10 ml/kg.bw, 20 ml/kg.bw) have proved that the effects of 5

ml/kg.bw IND on cachexia had reached a therapeutic plateau, therefore, 5 ml/kg.bw

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(1 mg/kg.bw) IND concentration was selected in this study. Mice in all three groups
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were administered i.p. once a day for 7 days. Body weight and tumor inoculation site
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for all mice were monitored weekly starting from Day 1. Food and water intake were

measured between 8:00 – 10:00 am daily. Tumor volume (V, cm3) was estimated from
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Day 4 onwards using the formula V=a*b2/2, with ‘a’ as length and ‘b’ as width. From
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the subcutaneous tumor block and the weight of mice, the anti-tumor rate (AR) was
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calculated, AR = (c - d) *100%/c, where ‘c’ was considered as the mean weight of

tumor in the control group, and ‘d’ stood for the mean weight of tumor in BJOE or

IND treatment groups. The expansion tablet counting method was employed to

estimate the extent of lung metastases (LM) [24], i.e., the neoplastic nodules of edges

on the lung tissue surface. Further, the LM inhibition rate (LMIR) = (e - f) *100%/e,
was calculated, where ‘e’ was the number of LM in the control group, and ‘f’ was the

amount of LM in BJOE or IND treatment group. Survival time for the remaining mice

(8 in each group) was recorded and elongation of survival time (EST) was calculated.

All mouse protocols were approved by the Ethics Committee in the Animal

Experimentation Department of Zhejiang Provincial Hospital of Traditional Chinese

Medicine, in accordance with the Association for Assessment and Accreditation of

Laboratory Animal Care International.

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Enzyme-linked immunosorbent assay (ELISA)

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Seven mice from each group were killed on day 31, with 1.5 ml of blood samples
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collected from the canthus vein of each mouse, before being sacrificed. Blood

samples were made to stand for 1 hour and then centrifuged for 10 minutes at 3000
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r/min. The serum was then stored at -70˚C. The levels of Tumor Necrosis Factor-α
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(TNF-α) and interleukin-6 (IL-6) were determined using ELISA kits, as per

manufacturer’s instructions.
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Bioinformatics analysis
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Gene expression profile

Total RNA was prepared from frozen tissue samples using TRIzol regent (Invitrogen,

Carlsbad, CA, USA) as per manufacturer’s instructions, and the Ambion Illumina

RNA Amplification Kit was selected to perform RNA amplification. Then, cDNA was
synthesized using reverse transcription with an oligo (dT) primer. After purification,

the cDNA was hybridized on mouse WG-6 BeadChip in a multi-step procedure as per

the manufacturer’s instructions. Fluorescent dye labeling was done in the

hybridization process, by mixing GEX-HBY hybridization solution and RNA-free

enzyme with SA-Cy3 (Fluorlink Cy3). The chips were then washed, dried, and

scanned on the Bead Array Reader, and raw data was generated using GenomeStudio.

The raw data was normalized with GenomeStudio using a Cubic Spline algorithm

capable of minimizing nonlinear transformation effects in data [25]. The

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normalization used quantiles of sample intensities to fit smoothing B-splines. Let

𝑖−0.5 𝑁𝑝𝑟𝑜𝑏𝑒𝑠

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𝑞𝑖 = (i = 1, 2, …, N) to be a vector of N quantiles, 𝑁 = max⁡(15, ), of
𝑁 100

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which Nprobes stood for the number of probes represented in a sample. After

normalization, probe correction was conducted for the perfect match and mismatch
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probes [26], and then the probe expression value [27] was explored. The preprocessed
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probe-level dataset in CEL formats were converted into expression measures, and

then screened using gene filter package [28]. Finally, a gene expression profile with
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30850 genes was obtained for further analyses.

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Identification of hub genes with potential prognostic impacts

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There were three groups of data in the gene expression profile, control, BJOE and
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IND treated group. These were then divided into two groups, BJOE group (BJOE

treatment vs. controls) and IND group (IND treatment vs. controls). To identify hub

genes with potential prognostic impacts, DEGs were first identified in the two groups,

and then PPI networks of DEGS were constructed utilizing STRING database, to

explore hub genes which might be potential biomarkers for lung cancer cachexia in
mice.

Detections of DEGs

In this paper, DEGs identified from the two groups were based on linear models for

microarray data (Limma) package [29]. The P values for all genes were converted into

-log10 form after being manipulated with t and F test. Linear fit, empirical Bayes

statistics and false discovery rate (FDR) correction were applied to the available data

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using Fit function [30]. Genes with threshold value of P < 0.05 and

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|log2FoldChange| >2 were regarded as DEGs.

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Construction of PPI network
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In this section, possible functional associations between DEGs were investigated

using the STRING database, which provided a comprehensive, quality-controlled

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collection of gene/protein associations for a large number of organisms on a global

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scale [31]. STRING could assess and integrate DEGs to obtain a confidence score for

all protein/gene interactions. Subsequently, these interactions were visualized by

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Cytoscape [32], a free software package for visualizing, modeling and analyzing the
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integration of bimolecular interaction networks with high-throughput expression data

and other molecular states.

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Topological analysis of PPI networks

One of the fundamental problems in network analysis is to determine the importance

of a particular vertex or an edge in a network [33]. Quantifying centrality and

connectivity helps identify portions of the network that may play interesting roles.
Research has revealed that topological centrality is effective for identifying essential

molecules in well-characterized interaction networks [34]. In order to understand the

functionality of complex systems of gene signatures in the PPI networks in two

conditions, the biological importance of genes was characterized using topological

analysis (degree) index. The degree quantifies the local topology of each gene by

summing up the number of its adjacent genes [35]. It gives a simple count of the

number of interactions in a given node. The genes at the top of the degree distribution

(≥ 95% quantile) were defined as hub genes in the significantly perturbed networks.

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The degree D (v) of a node v was defined as

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D (v )   a vj
j

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The relationship between the number of genes and degree distribution was also

studied. As the PPI networks in general are modular and scale-free, a fitting
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coefficient R2 of the power-law 𝑌 = 𝑎𝑋 𝑏 of the objective networks was then
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detected, which meant that the networks had power-law (or scale-free) degree

distributions [36,37]. The Network Analyzer 2.7 plugin in Cytoscape 3.1.0 was used
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for the evaluation of topological parameters.

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Statistical analysis
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The results were analyzed using Statistical Product and Service Solutions (SPSS,
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Chicago, IL, USA) [38]. The data was expressed as mean ± standard deviation. One

way ANOVA was then implemented to compare the differences between the groups.

In addition, 0.01 < P < 0.05 was considered to be a statistically significant difference,

and P < 0.01 was regarded as statistically significant.

Results

BJOE led to obvious improvement in cachexia status.

In preliminary experiments, it was found that there was an obvious change in the

physical status and amount of activity in mice 24 days after inoculation, including

anorexia, hypokinesis and subcutaneous tumor growth. The body weight decreased

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rapidly on the 24th day, and the mice entered into a state of cachexia, hence they were

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treated from Day 24 for 7 days. Right from the time they were inoculated to 24 days

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later, the food and water intake of the mice decreased persistently, and no significant

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differences in body weight were observed in all three groups. Post-treatment, in the
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BJOE group, the body weight, and food and water intake in mice showed no
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significant decline, while that in the control group decreased markedly. In the IND

groups, the decrease in body weight was similar to the control group. There was an
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obvious increase in food intake and water intake showed no significant decline. It was
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apparent that body weight improved more in the group on BJOE than those in the
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IND one. (Figure.1, Table.1).

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BJOE inhibited tumor growth and increased survival time in mice with cachexia.

After treatment, the tumor growth rate in mice was slower, and tumor weight and

pulmonary metastases were significantly less in the BJOE group as compared to the

control group. Also the anti-tumor rate was 22.5% and pulmonary metastasis
inhibition rate was 39.75% on day 7, and the survival time in mice was significantly

longer. Whereas, there was no obvious difference in tumor growth rate in the IND

group as compared to those from the control group. The average tumor weight was

more, pulmonary metastasis inhibition rate was 13.25% on day 7, and the survival

time in mice was significantly longer. On comparing BJOE and IND, there was a

more marked effect of BJOE on survival time extension, tumor growth rate and

pulmonary metastasis inhibition, than in the IND group (Figure.1, Table.1).

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BJOE lowered TNF- α and IL-6 in mice with cachexia.

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Cytokines including TNF-α, interleukin (IL)-1a, IL-1b, interferon-c, and IL-6 play an
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important role in the occurrence and development of cancer cachexia [39]. In this

experiment, both TNF-α and IL-6 in mice decreased significantly in the BJOE group
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than in the control group. The effects of IND were similar to BJOE; however,
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cytokine decrease was greater in mice treated with BJOE than with IND (Table.1).
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Nmd3, Bcl2, Nhp2l1 may be important drug targets for BJOE.

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The correlation between average signals for BJOE treatment to normal controls was
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similar to that of IND treatment in normal controls (Figure.2 & 3). Totally, 555 and

519 DEGs were identified for BJOE and IND groups, respectively, considering P <

0.05 and |log2FoldChange| >2. To explore the significance of gene interactions among

DEGs, PPI networks were constructed on the basis of the STRING database.

Simultaneously, the degree of distribution for all genes mapped to the PPI network

was displayed, as shown in Figure 4. There were 172 nodes and 251 edges (Figure 5)
in the BJOE group. Network analysis showed that the PPI network presented

scale-free properties and the degree of distribution followed a power law (𝑌 = 𝑎𝑋 𝑏 ,

where a = 67.05, b = -1.107) with the fitting coefficients R2 (R2 = 0.9985). A total of

138 DEGs were mapped to PPI networks in the IND group, with 177 resultant

interactions (Figure 6). The degree of distribution of PPI network in the IND group

also obeyed another power law, 𝑌 = 𝑎𝑋 𝑏 , where a = 60.91, b = -1.137 and R2 =

0.9868. These indicated that the two PPI networks possessed properties of small

world network.

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A degree centrality analysis was conducted, and 7 hub genes (Nmd3, Bcl2, Ftsj3,

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Rhob, Nhp2l1, Cct2 and Tfb1m) for the BJOE group and 6 hub genes (Bcl2, Frk,

Nmd3, Eps15, Myh2 and Nhp2l1) for the IND group were obtained. Interestingly,

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three common hub genes were found for the two groups – Nmd3, Bcl2, and Nhp2l1,
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which might play more important roles in Lewis lung carcinoma in mice than other

genes, and have greater potential to be prognostic markers of this tumor target
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treatment.
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Discussion
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In this study, it was found that BJOE was effective in treating lung cancer cachexia.

Furthermore, it exhibited greater superiority with respect to improvement in body

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weight, tumor growth and pulmonary metastasis inhibition, and survival extension

time compared to IND, which is commonly used in clinical practice. The function was

achieved through down-regulating the level of TNF-α and IL-6, and regulating the

relevant target genes in the downstream pathway. In this experiment, body weight,

food and water intake, tumor growth (tumor weight and tumor volume), pulmonary
metastasis, and survival time were compared in mice treated by BJOE, IND and

normal saline solution. BJOE significantly slowed down weight loss and tumor

growth, increased food and water intake, inhibited pulmonary metastasis, and

prolonged the survival time as compared to the control group.

IND is commonly used to treat patients with cancer. Ikawa et. al. had

demonstrated that Indometacin could antagonize prostanoids such as prostaglandin E

(2), which is considered as a biomarker for colorectal cancer [40]. Also IND

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decreased protein degradation by acting on ubiquitin-dependent proteases, reduced the

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levels of TNF-α and IL6, and alleviated muscle wasting in animals with cancer [41].

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A clinical study of patients with cancer cachexia indicated that IND suppressed

cyclooxygenase, improved energy homeostasis, and reduced chronic inflammation

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[42]. However, as a cyclooxygenase-inhibitor, IND had some typical side-effects,
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including gastrointestinal ulceration, blood coagulation disorders and anaphylaxis,

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which limited its application in clinical treatment. It has been demonstrated that BJOE

has significant use in the treatment of lung cancer, with prominent benefits and lesser
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adverse effects [43]. It could also induce cell cycle arrest and apoptosis via reactive
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oxygen species-mediated mitochondrial dysfunction in human lung cancer cells [44],

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but it has not been used so far for treatment of lung cancer cachexia. In this study, the

treatment effects of BJOE and IND on lung cancer cachexia in mice was compared,

and better results were observed in the group on BJOE as compared to those on IND.

Therefore, BJOE could be used as a candidate or auxiliary drug in the treatment of

lung cancer cachexia in the future.

 Excessive elaboration of pro-inflammatory cytokines such as IL-6 and TNF-α is

involved in cachexia-related hyper-catabolism [45]. IL-6 is a promising discovery in

the therapeutic strategy for attenuating cachexia progression through target adipose,

skeletal muscle, gut, and liver tissue. All of these factors affect patient recovery from

cachexia. TNF-α has a direct catabolic effect on skeletal muscles and causes muscle

wasting by the induction of the ubiquitin-proteasome system (UPS). TNF-α is

responsible for increasing gluconeogenesis, loss of adipose tissue and proteolysis,

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resulting in decrease in protein, lipid and glycogen synthesis in cancer cachexia

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[46,47]. In this study, BJOE obviously decreased TNF-α and IL-6 levels, thus

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illustrating that BJOE could decrease cancer cachexia to some extent, based on the

reduction of serum TNF-α and IL-6 levels.

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Successful treatment of diseases was commonly done through gene expression
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regulation and some important signal transmission. In order to further explore the
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treatment mechanism of BJOE for lung cancer cachexia, the influence of BJOE on

genes was analyzed, based on appropriate gene chips. In this study, 7 hub genes
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(Nmd3, Bcl2, Ftsj3, Rhob, Nhp2l1, Cct2 and Tfb1m) were found. Interestingly, Nmd3,
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Bcl2, and Nhp2l1 were also found in the IND treatment group, which meant that they
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were more important than other hub genes in the treatment of lung cancer cachexia.

Nmd3 (nonsense-mediated decay 3) is an adaptor for exporting 60S ribosomal subunit

from the nucleus, and binds to nascent 60S subunits while exporting receptor

chromosome region maintenance protein 1 (Crm1). This facilitates its passage

through the nuclear pore complex [48]. Moreover, Crm1 and Nmd3 have complex
functions in pathways that couple rRNA synthetic and processing engines, and the

dysregulated functions may have been responsible for the onset of cancer [49]. A

mutation in Nmd3 was found to be lethal in the absence of 5'-3' exoribonuclease 1

(XRN1), which encodes a major cytoplasmic exoribonuclease, also responsible for

mRNA turnover [50]. Bcl2 (B-cell CLL/lymphoma 2) family proteins are key

regulators of the cell apoptotic process [51]. It is predominantly located in

mitochondria, which is the central coordinator of both intracellular and extracellular

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signals, mediating cell death of caspase-dependent and caspase-independent [52,53]

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cell structures. Stolz et al reported that induction of mitochondrial apoptosis is

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required in the involvement of BCL-2 [54]. Furthermore, overexpression of Bcl2

blocks TNF-related apoptosis-inducing ligand (TRAIL), induced apoptosis in human

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lung cancer cells [55]. Besides, Bcl2 acted synergistically in the occurrence,
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development, invasion and metastasis of NSCLC [56,57], and the co-expression of

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survivin and Bcl2 may serve as a biomarker for predicting prognosis of lung cancer

[58]. Therefore, Bcl2 played a significant role in lung cancer and might be a potential
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biomarker for lung cancer target therapy. Nhp2l1 (NHP 2-like protein 1, fibrillarin)
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process pre-rRNA through the post-transcriptional modifications, 2’-O-methylation

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and pseudouridylation reaction, as the core member of the box C/D small nucleolar

RNP (snoRNP) complex in nucleolus, its dysregulation is frequently observed in

cancer [59]. It is also an essential regulator of myogenesis by preventing muscle

defects and stabilizing skeletal muscle development [60]. It thus has close relation

with weight loss in cancer cachexia. Also some new genes (Ftsj3, Rhob, Cct2 and
Tfb1m) were discovered in the BJOE group but not in the IND group, suggesting that

BJOE is superior to IND in the treatment of cancer cachexia. Cachexia may be

responsible for these key genes, but further study needs be carried out to verify this

inference.

Conclusions

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In conclusion, BJOE alleviated lung cancer cachexia and inhibited the tumor. The

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mechanism involved down-regulating inflammatory cytokines and regulating the

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expression of the relevant genes, mainly Nmd3, Bcl2, Ftsj3, Rhob, Nhp2l1, Cct2 and

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Tfb1m. This finding provides new insights into treatment of lung cancer cachexia.
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Acknowledgments
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We thank colleagues affiliated with our laboratory for their technical assistance and
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helpful comments.
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Compliance with ethical standards

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All mouse protocols were approved by the Ethics Committee in the Animal

Experimentation Department of Zhejiang Provincial Hospital of Traditional Chinese

Medicine, in accordance with the Association for Assessment and Accreditation of

Laboratory Animal Care International.

Declaration of interest

The authors have no conflict of interest.

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Table

Figures

Figure legends

Figure 1 Changes in body weight, food and water intake, tumor volume in

tumor-bearing mice. (A) body weight, (B) food intake, (C) water intake, and (D)

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tumor volume

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Figure 2 Fluorescently-labeled scan images of chips for control group (A), Brucea

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javanica oil emulsion group (B) and Indometacin group(C).
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Figure 3 Average signal strength for gene expression profile. A: Brucea javanica oil

emulsion treatment vs. normal controls (BJOE group); B: Indometacin treatment vs.
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normal controls (IND group).

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Figure 4 Distribution of genes in the PPI networks. A: Brucea javanica oil emulsion

treatment vs. normal controls (BJOE group); B: Indometacin treatment vs. normal
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controls (IND group).

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Figure 5 PPI networks for Brucea javanica oil emulsion treatment vs. normal controls.
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Nodes were genes and edges were the interactions among genes. The roseo nodes

represented hub genes with degree sequence ≥ 95%.

Figure 6 PPI networks for Indometacin treatment vs. normal controls. Nodes were

genes and edges were the interactions among genes. The roseo nodes represented hub

genes with degree sequence ≥ 95%.

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Table 1 Effects of BJOE and IND on mice with cachexia

Group control BJOE IND

Bodyweight, g 18.71±1.64 20.08±1.37* 18.88±2.23

Food intake, g 2.09±0.09 2.41±0.17* 2.34±0.10*

Water intake, ml 2.77±0.17 3.23±0.26* 3.26±0.35*

Tumor mass, g 1.88±0.62 1.47±0.53* 2.15±0.22

AR, % 0 22.05% -14.34%

Tumor volume, cm3 1.46±0.70 1.09±0.53 1.92±0.48

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NLM 11.85±3.18 7.14±2.68* 10.28±2.63

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LMIR, % 0 39.75 13.25

Survival time, day 36.00±2.37 45.57±4.47** 43.17±3.12**

TNF-α, fmol/ml 26.79±13.79

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8.50±6.02** 11.99±6.85*
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IL-6, pg/ml 200.94±99.57 83.99±48.72* 90.25±37.86*
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AR, NLM, LMIR, were abbreviations of anti-tumor rate, number of lung metastasis

and lung metastasis inhibition rate respectively. * P < 0.05 significantly different
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compared to control group. ** P < 0.01 significantly different compared to control

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group. Data are expressed as the mean ± SD.

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