Berberine chloride induced PPARƳ activation

mediated apoptosis resulted by activation of ROS

generation and COX-2 inhibition in HepG-2 cells

Dr. Hemanth Kumar Manikyam

Ph.D(BIOCHEM),Ph.D(CHEM), ONCOBIOLOGY, MBA (HEALTH CARE MANAGEMENT

Presented @

INTERNATIONAL CONFERENCE ON AYURVEDA TRADITIONAL MEDICINE AND MEDICINAL PLANTS

INTRODUCTION
 Berberis aristata
Taxon identifier 659592

Berberis aristata, also known as Indian barberry, "chutro"

or tree turmeric, is a shrub belonging to the
family Berberidaceae and the genus Berberis. The genus
comprises approximately 450-500 species of deciduous Fig 1
evergreen shrubs and is found in the temperate and sub- Kingdom : Plantae
tropical regions of Asia, Europe, and America. B. aristata is Unranked : Angiosperms
native to the Himalayas in India and in Nepal. It is also Unranked : Eudicots
naturally found in the wet zone of Sri Lanka. Order : renunculales
Famliy : Berberidaceae
Medicinal Uses :
Genus : Berberis
In India, B. aristata is used in traditional herbal medicine. Its
Species : B.aristata
stem, roots, and fruits are used in Ayurveda. A preparation
called rasaunt is prepared by boiling the bark of the root and
of the lower part of the stem in water. The solution is then
strained and evaporated until a semi-solid mass, rasaunt, is
obtained. It is mixed with either butter and alum, or with
opium and lime juice. The root bark contains the bitter
alkaloid berberine, which has been studied for its potential
pharmacological properties.
Fig 2
 Berberine Hydrochloride Fig 4
Berberine Fig 3

Pharmacology :
The pharmacologic actions of berberine include metabolic inhibition of
certain organisms, inhibition of bacterial enterotoxin formation, inhibition of
intestinal fluid accumulation and ion secretion, inhibition of smooth muscle
contraction, reduction of inflammation, platelet aggregation inhibition,
platelet count elevation in certain types of thrombocytopenia, stimulation of
bile and bilirubin secretion, and inhibition of ventricular tachyarrhythmias.

In addition, berberine may possess anti tumor promoting properties as

evidenced by inhibition of COX-2 transcription and N acetyltransferase
activity in colon and bladder cancer cell lines and transient, but marked,
inhibitory action on the growth of mouse sarcoma cells in culture.
 Parts Used :

Roots and Stems

Extraction Procedure of berberine hydrochloride :

Roots and stems are subjected to Microwave assisted

subcritical water extraction by continuous soxhlet
method had been used as mentioned by Hemanth Fig 4
kumar manikyam et al (2017).

Fig 5
1.PPAR GAMMA NUCLEAR RECEPTOR

PPAR Gamma is activated by prostaglandin D2 metabolite 15-deoxyÄ

prostaglandin J2 (15 d-PGJ2) and anti-diabetic drugs, resulting in down-regulation
of the expression of pro-inflammatory genes and inhibition of tumor cell growth.
PPPAR Gamma regulates lipid metabolism in adipose tissues.

2.CYCLOOXYEGNASE- 2
Cyclooxygenases are key enzymes in the prostaglandin synthesis. The inducible
isoenzyme cyclooxygenase-2 (COX-2) plays a pivotal role as a mediator of
inflammation. In addition, COX-2 expression is associated with carcinogenesis
most likely by modulating apoptosis (Dannenberg et al., 2001). With regard to its
latter function, COX-2 was shown to be upregulated in a variety of human cancers
including colon, gastric, oesophagus, pancreas and breast cancer, while
undetectable in most normal tissues (Eberhart et al., 1994; Subbaramaiah et al.,
1996; Ristima ki et al., 1997; Hida et al., 1998; Tucker et al., 1999; Zimmerman et
al., 1999)
 3. REACTIVE OXYGEN SPECIES

Constant generation of low levels of reactive oxygen species (ROS) and free radicals is a basic
feature of all living cells. Low levels of ROS play an essential role in signalling pathways,
whereas increased under oxidative stress, ROS activity result in damage to nucleic acids,
proteins and membrane lipids. Accumulation of ROS during oxidative stress is also associated
with aging, apoptosis or necrosis, and is implicated in pathological conditions such as;
vascular diseases, diabetes, renal ischemia, arteriosclerosis, pulmonary disorders,
inflammatory diseases, and cancer. Cellular activity of ROS is offset by antioxidants,
numerous repair systems, and replacement of damaged DNA. Probes for measuring
intracellular ROS levels provide important tools to study oxidative stress inducers and effects
of antioxidant therapies

4. CERAMIDE
One of the most studied roles of Ceramide pertains to its function as a proapoptotic
molecule. Apoptosis, or Type I programmed cell death, is essential for the maintenance of
normal cellular homeostasis and is an important physiological response to many forms of
cellular stress. Ceramide accumulation has been found following treatment of cells with a
number of apoptotic agents including ionizing radiation, UV light, TNF-
alpha, and chemotherapeutic agents. This suggests a role for Ceramide in the biological
responses of all these agents. Because of its apoptosis-inducing effects in cancer cells,
Ceramide has been termed the "tumor suppressor lipid". Several studies have attempted
to define further the specific role of Ceramide in the events of cell death and some
evidence suggests Ceramide functions upstream of the mitochondria in inducing apoptosis
Fig 6
PROTEIN TARGETS OF BERBERINE

Fig 7
 Proposed pathway
Fig 8
CAONICAL PATHWAY
PPAR ƳNULCEAR
Berberine RECEPTORS
NON CANONICAL PATHWAY
COX 2
Plasma membrane INHIBITION
potential

Apoptosis

Ceramide synthesis
Caspase cascade
activity
Mitochondria redox
enzymes
Bcl-2 down regulation

ROS
generation Bax/bak up regulation
Materials & Methods
HepG2 cell line culture
MTT Assay kit
PPAR GAMMA colorimetric assay kit
 PRSOTAGLANDIN E2 Assay Kit
ROS Assay kit

METHODS

1. HepG2 Cell Culture

HepG2 Cell lines were purchased from National Center for Cell Sciences, Pune, with
seeding density of 2.0 × 104 cells/well stored in liquid nitrogen for further testing
purpose.

Prior to the assay the test system Hep-G2, cells were propagated at °C in a gaseous
environment of 5% ± 1% carbon dioxide, in humid environment in tissue culture flasks
containing medium, Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, USA)
supplemented with 10% fetal bovine serum (Invitrogen, USA), and penicillin (100 units)
and streptomycin (100 μg) antibiotics (Invitrogen, USA) to obtain the subconfluence of
cells (70% to 90% confluent).
2. Cell Seeding for Cytotoxicity Assessment MTT Assay
Cell monolayer was rinsed with PBS and aspirated off PBS and cells were trypsinized with
0.25% Trypsin with 0.2 g/L EDTA in tissue culture flask at °C until the cells detached and
floated. DMEM with 10% FBS was added into the flask to flush out the cells and centrifuged
at 900 rpm for 5 minutes. Cells were resuspended in DMEM medium and cell suspension was
subjected for the cell count and viability to determine cell number per mL. Cell number was
adjusted to 2 × 105 cells/mL and 0.1 mL of the adjusted cells was seeded in each well of 96-
well cell culture plates. Frequent mixing was done during the seeding, to achieve a uniform
cell suspension for plating the cells per well. Plates were designated to indicate its contents
and date of experiment. Plates were incubated at °C for  hrs in gaseous environment of 5% ±
1% carbon dioxide.
After  24 hrs of incubation the cells were exposed to different concentrations of test items,
by replacing the spent medium with 100 μL of different concentrations of the test items
solution and incubated for  hrs at °C in gaseous environment of % carbon dioxide. Positive,
negative control and blank were dispensed in the designated wells and incubated for  hrs
at °C in gaseous environment of % carbon dioxide. At the end of the  hrs incubation medium
with test item/positive control was removed and cells were incubated for 4 hrs with 20 μL of
MTT 5 mg/ mL solution. After 4 hours of incubation formazan crystals formed by
mitochondrial reduction of MTT were solubilized by adding 150 μL of DMSO. Absorbance was
read at 570 nm after 10 min incubation with vortexing.
 2.1 Data Analysis

A decrease in the number of living cells results in a decrease in the metabolic activity in
the sample. This decrease directly correlates to the amount of formazan formed as
monitored by optical density at 570nm. Percent Viability will be calculated using the
below formula.

% Viability = 100 (O.D Test Item / O.D of Control)

% Activity = 100 - % Viability
 3. PPAR-γ Colorimetric Cell-Based ELISA Kit was purchased from CytoGlow™ Catalog #:
CB5585
Fig 9
Fig 10
Protocol
4. In vitro COX inhibition assay
The ability of Berberine to inhibit ovine COX-1 and COX-2 was determined using an enzyme
immunoassay (EIA) kit (catalog no. 560101; Cayman Chemical Co., Ann Arbor, MI, USA). COX catalyzes
the first step in the biosynthesis of AA to PGH 2. PGF2α, produced from PGH2 by reduction with stannous
chloride, was measured by EIA (ACE™ competitive EIA, Cayman Chemical, Ann Arbor, MI, USA). Briefly,
to a series of supplied reaction buffer solutions [960 μL 0.1 M Tris-HCl (pH 8.0) containing 5 mM EDTA
and 2 mM phenol] with either COX-1 or COX-2 (10 μL) enzyme in the presence of heme (10 μL), 10 μL
of various concentrations of test drug solutions (1, 10, or 100 μM in a final volume of 1 μL) were
added. These solutions were incubated for 5 min at 37°C and subsequently 10 μL AA solution (100 μL)
was added. The COX reaction was stopped by the addition of 50 μL 1 M HCl after 2 min. Then 100 μL of
stannous chloride was added to produce PGF2α, which was measured by EIA. This assay is based on the
competition between PGs and a PG- acetylcholinesterase conjugate (PG tracer) for a limited amount of
PG antiserum. The amount of PG tracer that is able to bind to the PG antiserum is inversely
proportional to the concentration of PGs in the wells since the concentration of the PG tracer is held at
a constant while the concentration of PGs varies. The specific antiserum-PG complex bound to a
mouse anti-rabbit IgG that had been previously attached to the well. The plate was washed to remove
any unbound reagents and 200 μl Ellman’s reagent, which contains the substrate to acetylcholine
esterase, was added to the well. The product of this enzymatic reaction generates a distinct yellow
color that absorbs at 406 nm. The intensity of this color, determined by spectrophotometry, is
proportional to the amount of PG tracer bound to the well, which is inversely proportional to the
amount of PGs present in the well during the incubation. Percent inhibition was calculated by the
comparison of the compounds treated to the various control incubations.
5. Measurement of PGE2
HepG2 cells were plated at a density of 2.5×105/ml cells in a 24-well plate with 1 ml of
culture medium per well and cultured overnight. The cells were pre-incubated for 2 h
with various doses of Berberine and stimulated for 24 h with 100 ng/ml LPS. The cell
culture supernatants were collected immediately following treatment and centrifuged
at 1,000 × g for 15 min to remove the particulate matter. PGE2 was determined using
an enzyme immunoassay (EIA) kit (catalog no. ADI-900-001, Enzo Life Sciences,
Switzerland). The medium and PGE2 EIA conjugate was added to a 96-well plate pre-
coated with goat anti-mouse IgG and left to react for 2 h, followed by a final wash to
remove any unbound antibody-enzyme reagent. A substrate solution was added and
the intensity of the color produced was measured at 405 nm (correction wavelength
set at 570–590 nm).

5.1 PRINCIPLE
Prostaglandin E2 (PGE2) is a primary product of arachidonic metabolism and is
synthesized via the Cyclooxygenases (COX) and prostaglandin synthase pathways.
PGE2 production is a commonly used method for the detection of COX-1 and COX-2
modulation and prostaglandin synthases. The HTRF® PGE2 assay is a highly sensitive
method for quantifying PGE2 either in cell supernatants or directly in the presence of
whole cells. Our reagents have simplified protocols requiring only two reagent
additions. These protocols are easily miniaturized and amenable to automation and
HTS.
RESULTS
% activity of berberine in HepG-2 cells after 24 hrs incubation- EC50-0.214 mg/ml

Average Percent Average Percent Activity of

Concentration in Activity of berberine Concentration in Positive Control (SLS)
mg/ml Percentage

HEPG2 HEPG2

0.500 85.085 5.000 100.092

0.167 45.384
2.500 99.610

0.056 19.290
1.250 99.037
0.019 -1.547
0.625 98.716
0.006 5.816
0.313 98.097
0.002 4.136

Table 2
Table 1

Graph 1
 C LPS LPS+IM 0.01 0.1 1 10 100 µmol/l
Graph 2

Inhibitory effects of Berberine on LPS‑ induced PGE2

production in HepG2 cells.

C –Control, LPS – edotoxin lipopolysachharides, IM- Indomethacin positive

control
Inhibitory effects of berberine Hcl in vitro COX-2 enzyme activity

Group PGF2 alpha pg/ml Inhibitory rate %

COX-2 inhibitor tubes 0.9+/-0.1
COX-2 100% Initial 135.5+/-23.9
activity tubes
NS umol/l 40.2+/-5.8 75.21
10
Berberine Hcl umol/l
100 41.2+/-8.1 76.4
10 120+/-5.6 21.54
1 145.5+/-6.9 0.79

Table 3
0 1 6 12 18 24 Time Hr

COX2

PPAR GAMMA

bax

bak

bcl-2

Effect of berberine hydrochloride on levels of COX-2, PPAR GAMMA, bax,

bak, bcl-2 proteins in HepG2 cells indicated at time

Figure 11 WESTERN BLOTT ANALYSIS

 Discussion

Berberine a PPAR Gamma agonist and COX 2 inhibitor induces HepG2 cell apoptosis via
Ceramide ROS signalling Cascade by upregulating Bax and Bak signalling proteins and down
regulating Bcl-2 proteins triggering Caspase 3 cascade. Berberine has shown 0.2140 mg/ml EC 50

Berberine acts as PPAR Gamma agonist which is a nuclear receptor that enhance lipid oxidation
thus promoting acetyl esterase activity in mitochondria, peroxisomes, lysomes and of Golgi
complex and endoplasmic reticulum leading to synthesis of sphingolipids. Over expression of
sphingolipids triggers Ceramide synthesis in plasma membrane . Ceramide increases Calcium
out flux from mitochondria leading to more ROS generation that triggers Bax and Bak leading to
Caspase cascade activity and apoptosis.
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