BETA SITOSTEROL STIMULATE PHAGOCYTOSIS IN HEPG2 CELL LINE TRANSFECTED WITH HEPATITIS B SURFACE ANTIGEN

www.wjpr.net Vol 6, Issue 4, 2017. ₄₁

BETA-SITOSTEROL STIMULATE PHAGOCYTOSIS IN HEPG2

CELL LINE TRANSFECTED WITH HEPATITIS B SURFACE

ANTIGEN

Ammar M. Lubeiny1-2*, Wuyun Li2, Xu Lin2 and Elshaikh A. Elobied3

1

Neelain Institute for Medical Research, Faculty of Medicine, Al-Neelain University,

Khartoum, Sudan.

2

Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical

University Fuzhou, China.

3

Research and Grant Unit, Ahfad University for Women, Khartoum, Sudan.

ABSTRACT

Hepatocellular carcinoma (HCC) has a range of risk factors and

Hepatitis B Virus infection considers as one of these factors which

lead to develop HCC. This type of carcinoma is still classified as a

complex disease due to many risk factors and the patients with HCC

diagnosed always at advance stage. The surgery and transplantation

are still the most effective treatment while the response to

chemotherapy and radiotherapy has poor effect. Our aim is to study

the effect of β-Sitosterol, which is found in many plants on HBsAg

and on hepatocellular carcinoma. To address this issue, Huh7,

HepG2.2.15( harbouring four copies of HBV DNA ) and HepG2 cell

lines were used. The Cell viability assay, Enzyme – Linked

Immunosorbent Assay, and Gene Expression were used to evaluate the effect of

β-Sitosterol. The result of Gene Ontology shows it is effect on HepG2-HBsAg by stimulating

the phagocytosis we conclude β-Sitosterol has a positive regulation of phagocytosis to

HepG2 Transfected with HBsAg this process could be help full to eliminate the dead cells

caused by the apoptotic effect of β-Sitosterol on hepatocellular carcinoma cell infected with

hepatitis B virus.

KEYWORDS: Beta- sitosterol, phagocytosis, HCC, Hepatitis B virus.

Volume 6, Issue 4, 41-51. Research Article ISSN 2277– 7105

*Corresponding Author

Dr. Ammar M. Lubeiny

Neelain Institute for Medical

Research, Faculty of

Medicine, Al-Neelain

University, Khartoum,

Sudan.

Article Received on 22 Jan. 2017,

Revised on 12 Feb. 2017, Accepted on 05 March. 2017

INTRODUCTION

The poor prognosis of HCC due to late and advance stage and a wide range factors

classified it as complex disease. Moreover cannot responsive to standard chemotherapy and

resist to radiotherapy while in the most cases the surgical and liver transplantation remains

only the choice for treatment. There for the development of novel effective treatment is

prime important.[1] The recent studies using a combination of different technologies, like

genomics, proteomics, metabolomics, MicroRNA studies, and Bioinformatics, are

providing new understanding of the gene expression and protein profiles to develop novel

cancer therapeutics .[2] A 30% of the global populations are infected with HBV and they are

at risk of developing liver fibrosis, HCC, and other illness.[3, 4, and 5] In this study, we used

DNA microarray to evaluate the effect of Beta sitosterol on Hepatoma cell HepG2

transfected with HBsAg. The HBsurface (HBs) proteins, consist of large, middle and small

(SHBs) (LHBs, MHBs, SHBs) proteins and it are possibly distinguished by their different

domains and glycosylation status.[6] The carboxyl terminal domain containing SHBs is

present in all surface-proteins, preS1 N-terminal extension only in LHBs, preS2 in LHBs

and MHBs.[7]. These three forms of HBV surface proteins represent HBsAg.[8] and

synthesized in the endoplasmic reticulum and could lead to the situation that amounts of

protein produced exceed those required for virion assembly. Excess surface proteins

undergo multimerization resulting in their budding from the ER/Golgi compartment as both

non-infectious spherical and filamentous sub viral particles (SVP) or as versions.[9] The

SVPs typically outnumber the virions, could be components of circulating immune

complexes.[10], and may induce immune tolerance by a mechanism of “viral apoptotic -like

mimicry” [11].

The domains of HBV surface proteins are S, preS2 domain is 55 aa long, and

preS1. PreS1 contains 108, 118, or 119 amino acids (aa), depending on the genotype, and

S-domain (S) PreS extensions of LHBs and MHBs possess certain features that are very

important for HBV infectivity such as binding the nucleocapsid during virus envelopment [9]

HBsAg could be involved in the arrangement, of the immune responses, especially in

disturbances of the appropriate immune responses. [12, 13, 14 and 15]. The HCC carcinogenesis

caused by HBV has been studied and the relations with HBV were proof as a multiple

factors process that includes a direct mechanism involving viral protein, indirect

mechanisms through the chronic inflammation, and the integration of HBV DNA.[16] The

direct mechanism of the viral protein reported in the HBx gene.[17, 18] and PreS2 gene.[19]

based on a transgenic mouse model and force expression model of the cell lines act as

mitogen-activated protein kinase (MAPK), which is involved in cell proliferation.[20] Moreover,

PreS2 protein accumulates in the endoplasmic reticulum (ER) of hepatocytes, and DNA

injury is caused in the cell by ER stress [21, 22] we subjected the Hepatoma cell lines Huh7,

HepG2 and HepG2.2.215 to β-Sitosterol to evaluate it is an effect of this compound which

can be found in many plants it will be important to evaluate its function on HBV and HCC.

Materials

β-Sitosterol (C29H50O) (Sigma Aldrich analytical standard) prepared by dissolved

0.995mg β-Sitosterol in 10 ml Ethanol HepG2.2.15 cells (CRL-11997; ATCC) harboring

four copies of HBV-DNA. HepG2 from American Type Culture Collection (ATCC). Cell

counting kit 8 CCK8, DOjinDO laboratories, Japan. G418 Geneticin anti-biotic (Invitrogen)

Method

Cell culture HepG2.2.15, Huh 7and HepG2 Cell lines were cultured in Dulbecco's

Modified Eagle Medium (DMEM) (Invitrogen) with 10%FBS (Fetal Bovine Serum) from

Gibco® and G418 Geneticin (Invitrogen) at 380 μg/ml added to HepG2.2.15

Cell viability assay: Cell Counting Kit 8 (CCK8, DOjinDO laboratories, Japan) was used

for the Cell viability determination. 8x103 cells from Huh7 HepG2 and HepG2.2.15were

counted (Count star Biotech Automated Cell Counter Shanghai) and cultured in 100 µl

DMEM with 10% FBS in 96 wells plate for 24 hours, β-Sitosterol added in different

concentration from 0.0, 60, 120, 240 μM . Incubated for 24 hours to determine the cell

viability in selected time. One hour before measuring the viability of the cells, The DMEM

were changed with 100 µl of 1:10 CCK-8 solution and DMEM and incubated for one hour

at 37°C in 5% CO2 incubator. Then we measured the absorbance at 450 nm using a micro

plate reader (ELx800 Absorbance Reader). Care was taken not to introduce bubbles to the

wells during changing the medium, since they interfere with the O.D. reading.

Enzyme – Linked Immunosorbent Assay (ELISA).

Detection of Hepatitis B Surface Ag: We incubate the HepG2.2.15 cells in plate till a

90% confluence was reached .Then β-Sitosterol in ethanol at 60 μM concentration were

added and incubated for 24 hours at 37°C in 5% CO2 incubator. the reagents kit used from

Wantai Beijing China and we followed the company protocol. One hundred µl taken from

control were added. Then 50μl from diluents were added to all the wells, the plate were shook slowly, cover and incubated for one hour at 37°C in 5% CO2 incubator. 50μl of

conjugate added and incubated for 30 minutes at 37°C in 5% CO2 incubator. The reagent

was decanted and the plate was tapped to remove any reagent from the wells. Cells were

washed by adding wash solution four times; incubated after addition of wash solution for

two minutes each time. Then 50μl of Chromogen A and 50μl Chromogen B solution were

added incubated for 30 minutes at 37°C in 5% CO2 incubator. We stopped the reaction by

adding 50μl from the stop solution and read at 450nM. Then we measured the absorbance at

450 nm using a microplate reader (ELx800 AbsorbanceReader). Care was taken not to

introduce bubbles to the wells during changing the medium

Plasmid construction: Plasmids included HBs cDNA, were constructed and used as

templates for polymerase chain reaction (PCR) amplification. PCR-generated HBx genes

were inserted into the KpnI and XhoI sites of the pcDNA3.1/Hygro (+) plasmid (V870–20,

Invitrogen), the resultant vectors were called pcDNAhygro+- HBs the primers used for PCR

amplification is shown as followed:

S-hygro (+) cloneF: 5’ - CGGGGTACC GCC ACC ATG GAGAACATCGC ATCAGG -3’

S-hygro (+) cloneR: 5’ - CCGCTCGAG CTA CTTGTCGTCA TCGTCTTTGT

AGTCAATGTA TACCCAAAGA-3’

Transfection: HepG2cell line were cultured in DMEM supplemented with 10% FBS in 10

cm plate till a 90% confluence reached then the 2ml trypsin were added for 3mints and

removed 3ml of DMEM were added, HepG2 cells were counted and seeded in 6-wells plate

at the density of 5× 105 per well 24 hours before transfection. The cells would reach 90%

confluent and the medium changed to DMEM without FBS and incubated for one hour

before transfection. Plasmids were transected into HepG2 cells using lipofectamine 3000

(Invitrogen, Carlsbad, CA) prepared in two test tubes one as reference containing 2.87 µl

pcDNAhygro+- plasmid and Salmon sperm DNA 12.4 µl,P3000™ 4 µl, Opti-MEM 80.73

µl the total amount 100.00 µl and the second tube PcDNAhygro+- HBs 2.56 µl , Salmon

sperm DNA 0.0 µl, P3000™ 4µl, Opti-MEM 93.44 µl the total amount 100.00 µl , In

another two tubes we prepared lipofectamine as in table 1 five minutes before transfection

we added the tubes of lipofectamine with the tubes containing PcDNAhygro+- HBs and

pcDNAhygro+- plasmid respectively. And transfer it to HepG2 cells in 10 cm plate. 24

the medium with the addition of Hygromycin 800μg/ml till reached the clear clone, and then

we picked the clones into 24 wells plate after the cells propagated, the cells were passage

into 12, 6 wells, 6 cm, and 10cm plate and propagated.

Table – 1 two tubes prepare with lipofectamine 3000

Component Amount

Lipo3000 3µl

Opti-MEM 97µl

Total amount 100µl

Gene chip

After transfection the HepG2-HBsAg, were cultured in DMEM with10% FBS in 10 cm

plates till reached a 90%confulence .Then the 60 μM of β-sitosterol were added and

incubated at 37 °C, 5% CO2 for 24 hours then the DMEM removed and 1.5 ml of Trizol

(Invitrogen) added to each plate We used Affymetrix gene chip and Agilent gene chip.

Total RNA quality control, mark, chip hybridization, washing, staining and scanning, and

data extraction and analysis procedure as company protocol. Affymetrix micro array for

detection of mRNA expression profiles of total RNA samples as the starting, and

biotin-labeled in vitro amplification, markAmbion #1792 cRNA expansion tags Kit. Data

extraction and analysis software using the AGCC (Affymetrix® Gene Chip® Command

Console® Software) chip . Fluorescent Scanned image is saved as DAT files and analyzed

with KEGG PATHWAY, P ID, PANTHER, REACTOME, BIOCART, BIO CYCL

software.

RESULT

Cell viability: Cell viability: Cell viability by CCK8 result shows decreases in viable cells

related to concentration of β-Sitosterol in three cell lines In previous study we proofed that β-sitosterol has effect on HepG 2 and HepG2.2.15 cell lines by reducing viable cells .It is

0 0.2 0.4 0.6 0.8

0 60 120 240

Figure 1 shows the viable cells in Huh7 Cell line y axis represented the number of

viable cell, x axis represented the concentration of Sitosterol column 1- No

β-Sitosterol were added, column 2- 60 μM β-β-Sitosterol , column 3-120 μM β-β-Sitosterol,

column 4- 240 μM β-itosterol

Effect of β-Sitosterol on HBsAg

The optical reading (OD) was indicating that 60 μM of β-sitosterol has an effect on

HepG2.2.15by reducing the total amount of HBsAg. as shown in figure 2.

Figure- 2 ELISA test for HBsAg quantification 1 is a reference shows the amount of

HBsAg in HepG2.2.15 Column 2shows the amount of HBsAg in HepG2215with 60 μM

beta-sitosterol this figure shows the decreased in HBsAg amount in cells treated with

beta-sitosterol

Gene Expression

Figure 3 Electrophoresis for quantification of the protein in the samples for

microarray test Lane 6 HepG2 HBsAG, Lane10 HepG2- PcDNA-hygro treated with

β-sitosterol, M Lane Hela Cell Control RNA

Figure 4 - Log-log scatter gives a visual comparison of gene level variation between

two cell lines. S6 refer to HepG2-HBsAg, the S10 is HepG2 cell line as reference both

the cell lines were treated with β-sitosterol red color represented up regulation of

genes, while green color down regulation and black equal expression in both reference

and sample

Gene Ontology

The result of Gene Ontology (GO) biological process increased the phagocytosis and

activation c3 and c5 pathways and increasing cell adhesion the –log (corrected p-value)

shows there are increased expression on several genes as shown in figure 7 and enrichment

Figure 5 Gene Ontology (GO) biological process increased in several genes involve in

biological process in HepG2-HBsAg

DISCUSSION

Phagocytosis is the way toward taking in microorganisms, parasites, dead cells, and foreign

debris by a cell [23].When β-sitosterol add to HepG2-HBsAg stimulate the phagocytosis by

positive regulation of phagocytosis/GO: 0050766 in HepG2cell line our previous study

showed β-sitosterol has an effect on HepG2-HBx by inducing the extrinsic apoptotic

signaling pathway more over ; it has senescence inducing effect to HepG2-HBSP, This

herbal compound (β-sitosterol) has anti-proliferative effect on Huh7cell line and its effect

on HepG2 , HepG.2.2.15shown in previous study. The extensive study by DNA microarray

and Gene Ontology studies under the biological process revealed that β-sitosterol can

stimulate the phagocytosis in HepG2-HBsAg .The important of the Phagocytosis of dying

cells is a major process that be the final stage of cell death in a tissue under basal

conditions, in a diseased tissue like cancer or after anticancer therapies.[24] The cluster of

differentiation (CD47) is a commonly expressed molecule on all cancers. Its function to

block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis

and elimination. CD47 is therefore a validated target for cancer therapies.[25] Studies from

the I.L.W. laboratory have shown that malignant cancers up-regulate the “don’t eat me”

signal CD47, presumably in their progression to allow escape from endogenous “eat me”

signals that were induced as part of apoptosis and programmed cell removal.[25, 26, 27and 28].

Treatment of Caski and HeLa cells with β-sitosterol significantly suppressed the expression

of genes involved in cell proliferation and oncogenic transformation, while augmenting the

expression of genes involved in apoptosis and tumor suppression. Ultrastuctural

anti-proliferative and anti-cancer activity of this natural product isolated from traditional

Chinese herbs.[29] We believe that β-sitosterol can stimulate the phagocytosis in

hepatocellular carcinoma infected with Hepatitis B Virus and by this process can eliminate

the defragmented and dead cells which occurs due to apoptotic effect of β-sitosterol on

hepatocellular carcinoma and HBV.

CONCLUSION

β-sitosterol has anti-proliferation effect on HCC cell lines Huh7, HepG2and HepG2.2.15

and stimulate the positive regulation of phagocytosis process in HepG2.2.15 by direct effect

on HBsAg. Therefore β-sitosterol has great effect on hepatitis B Virus replication and this is

the first study to evaluate the effect of β-sitosterol on HepG2-HBsAg as for our knowledge

and could open new areas of research on hepatitis B virus pathogenesis.

ACKNOWLEDGEMENT

This work done by the scholar position from the key laboratory of ministry of education for

gastrointestinal cancer Fujian medical university China.

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