Transcript Vero Cell Line

6th WORLD CONGRESS ON BIOTECHNOLOGY
Quercetin enhances the effect of Adriamycin in
human hepatocellular carcinoma (HepG2) cell lines
Dr.R. VENKATESWARI, M.Sc., Ph.D.
DEPARTMENT OF MEDICAL BIOCHEMISTRY
Dr. ALM PG INSTITUTE OF BASIC MEDICAL SCIENCES
UNIVERSITY OF MADRAS
TARAMANI, CHENNAI – 600 113, INDIA
INTRODUCTION
 Cancer is a group of diseases characterized by uncontrolled growth and spread of
abnormal cells.
 Cancer is caused by both external factors (tobacco, infectious organisms,
chemicals, and radiation) and internal factors (inherited mutations, hormones,
immune conditions, and mutations that occur from metabolism).
 These causal factors may act together or in sequence to initiate or promote
carcinogenesis (The American Cancer Society, 2008).
 If the spread (metastasis) is not controlled, it can result in death.
 Cancer is treated with surgery, radiation, chemotherapy, hormone therapy,
biological therapy and targeted therapy (Cancer, facts & figures, 2010).
 Primary liver cancer also called hepatocellular carcinoma or hepatoma may be the
most common cancer worldwide. It occurs with great frequency in Asia and Africa
and is becoming more common in the United States as a complication of chronic
Hepatitis B viral infection (Kumar et al., 2003).
 Hepatocellular carcinoma (HCC, also called malignant hepatoma) is the most
common type of liver cancer, most cases are secondary to either a viral hepatitis
infection (hepatitis B or C) or cirrhosis (Kumar et al., 2003) (alcoholism being the
most common cause of hepatic cirrhosis) (Parkin et al., 2001), and occurs more often
in men than women usually seen in the age group of 50 - 60.
 In countries where hepatitis is not endemic, most malignant cancers in the liver are
not primary HCC but metastasis (spread) of cancer from elsewhere in the body, e.g.,
the colon.
CAUSATIVE AGENTS OF
LIVER CANCER
HEPATITIS B
VIRUS
HEPATITIS C
VIRUS
ALCOHOL
LIVER CANCER
AFLATOXIN
Early detection
 Screening for liver cancer for high-risk persons (for example, those chronically
infected with HBV or HCV) are done with ultrasound or blood tests but has not been
proven to improve survival.
 At present, the best strategy to reduce the burden of cancer is the adoption of
preventive measures, including vaccination against HBV and the avoidance of highrisk behaviors such as intravenous drug use and alcohol abuse.
Diagnosis of Liver Cancer
 There is no reliable or accurate screening blood test for liver cancer. The most widely
used biochemical blood test is alpha-fetoprotein (AFP), which is a protein normally
made by the immature liver cells in the fetus and marker enzymes.
EPIDEMOLOGY AND PREVALENCE

Cancer affects people at all ages with the risk for most types increasing with
age (Parkin et al., 2001). In 2007, cancer caused about 13% of all human deaths
worldwide (7.9 million) and the rates are rising as more people live to an old age
and as mass lifestyle changes occur in the developing world (Jemal et al., 2011).

Cancers are caused by abnormalities in the genetic material of the
transformed cells (Saurabh Shukla et al., 2010), which may be due to the effects of
carcinogens, such as tobacco smoke, radiation, chemicals, or infectious agents.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in men
and the eighth most common cancer in women worldwide (Jelic, 2010, Okuda,
2000; Bruix, 2002), resulting in more than 1 million patients and over 260,000
deaths per year (Liu et al., 2006).

The incidence of hepatocellular carcinoma is increasing in many countries. The
estimated number of new cases annually is over 500,000, and the yearly incidence
comprises between 2.5 and 7% of patients with liver cirrhosis, and increased
prevalence of hepatitis C virus (HCV) infection, in most industrialized countries
(Taylor-Robinson et al., 1997; Deuffic et al., 1998).

The incidence varies between different geographic areas, being higher in
developing areas; males are predominantly affected (Montalto, 2002). The frequency
of liver cancer is high among Asians due to chronic hepatitis B infection and due to
hepatitis C infection and alcohol abuse in Japan, North America and Europe.
CHEMOTHERAPY

Chemotherapy is used as one of the most preferred ways of treating cancer.
It basically uses cytotoxic or anti cancer drugs to eradicate the cancer afflicted
cells. The drugs travel through the blood and reach the cancerous cells and
destroy them.
Cancer can occur at various parts of the human body and the chemotherapy
drugs need to be administered accordingly.

The most prominent Liver cancer chemotherapy drugs include Cisplatin,
Adriamycin (doxorubicin), Methotrexate and 5FU (fluorouracil) etc.

Like all chemotherapy drugs, the Liver cancer chemotherapy drugs also
come with their side effects on the body.

Adriamycin (ADR) is an anthracycline antibiotic which blocks
RNA and DNA synthesis equally. Cells in S-phase are most
sensitive to the drug.

The drug has two main mechanisms by which it causes cell death:

Intercalation: Adriamycin intercalates between adjacent nucleotides
along the DNA forming a tight DNA-drug interaction. This interaction
disrupts DNA synthesis and transcription.

Enzyme inhibition: Adriamycin binds and inhibits topoisomerase II, a
key enzyme involved in DNA synthesis.

Metabolism of the drug also generates oxygen free radicals which
damage DNA and prevents DNA synthesis.

The cardiotoxicity that results from administering doxorubicin is thought
to result primarily from the generation of damaging free oxygen radicals
but might be partly due to the inhibition of topoisomerase II. The free
oxygen radicals cause the peroxidation of lipid membranes and inhibit
mitochondrial respiration.
QUERCETIN
 Flavonoids are a large family of phenolic compounds or polyphenols with wide
therapeutic applications [Middleton E, 2000]. Quercetin is one of the most widely
spread naturally occurring flavonoids, found in onions, garlic, cabbage, leek,
broccoli, apples, blueberries, tea and red wine [Manach, 2004 ]. It is known that
quercetin may exhibit anti-oxidant properties due to its chemical structure,
particularly
the
presence
and
location
substitutions [Hardwood M, 2007].
Structure of Quercetin
of
the
hydroxyl
(-OH)
VEGETABLES
ONIONS
APPLES
GRAPES
BERRIES
GREEN TEA
Quercetin, a ubiquitous bioactive flavonoid, have been reported

to induce cell growth inhibition and apoptosis in a variety of cancer cells (Di
Carlo, 1999).

can inhibit the proliferation of cancer cells (Choi, 2001; Ong, 2004).

cause cell cycle arrests such as G2/M arrest or G1 arrest in different cell
types (Choi, 2001; Jeong, 1999).

quercetin may be a potential chemopreventive or therapeutic agent in
hepatocarcinoma cells (Granado-Serrano, 2006).

quercetin-mediated apoptosis may result from the induction of stress
proteins, disruption of microtubules and mitochondrial release of cytochrome c,
and activation of caspases (Ong, 2004; Wang, 1999).
AIM AND SCOPE OF THE PRESENT STUDY

During the last few decades, cancer research has focused on the idea that cancer
is caused by genetic alterations and that this disease can be treated by reversing or
targeting these alterations (Miguel López-Lázaro,2010).

Adriamycin is commonly used as a first line therapy for HCC (Julien Edeline et
al., 2009). Some chemotherapy drugs work by strongly promoting oxidation
especially the class of chemotherapy drugs called anthracyclines (Adriamycin and
epirubicin)
 Scientific evidence suggests that combining certain chemotherapy treatments with
certain antioxidants at specific dosages can help improve drug effectiveness or reduce
the severity of side effects (Perumal and Shanthi, 2005).
 Some of these antioxidants have been found to be useful for restoring the natural
antioxidants in the body, which are often depleted after the completion of
chemotherapy.

IN VITRO STUDIES
 Cell lines are widely used in many aspects of laboratory research particularly
as in vitro models in cancer research. They have a number of advantages; for
instance, they are easy to handle and represent an unlimited self-replicating
source that can be grown in almost infinite quantities.
 Though animal model studies are the end stage before trying in humans to
understand the efficacy of the drug in biological milieu, use of them for
screening a large number of compounds becomes too expensive.
 With increasing demands on drugs for various diseases and to overcome the
resistance these drugs develop in due course, we need a faster method to
delineate and select the candidate drugs that can be successfully derived. In
vitro models appeared a practical alternative to this.
 The aim of the in vitro study was to investigate the hepatoprotective effect of quercetin
along with adriamycin on the apoptotic pathway in a human hepatoma cell line (HepG2).
HepG2 Cell Line

HepG2 is a human liver carcinoma cell line (Hepatocellular carcinoma, human). HepG2
is a perpetual cell line which was derived from the liver tissue of a 15 year old Caucasian
American male with a well differentiated hepatocellular carcinoma. HepG2 cells are a suitable
in vitro model system for the study of human hepatocytes.
Vero Cell Line

Vero cells are are one of the more commonly used mammalian continuous cell lines in
microbiology, and molecular and cell biology research. The Vero lineage was isolated from
kidney epithelial cells extracted from an African green monkey (Chlorocebus sp.; formerly
called Cercopithecus aethiops) (Yasumura and Kawakita, 1963). The original cell line was
named "Vero" after an abbreviation of "Verda Reno", which meaans "green kidney" in
esperanto, while "vero" itself means "truth" also in Esperanto (Shimizu B 1993).

The cell lines were obtained from the Department of Biotechnology, National Centre
for Cell Sciences (NCCS), Pune, India.
The cell lines were maintained and the
experiments were carried out at the animal tissue culture laboratory, Lifeteck Research
Laboratories, Vadapalani, Chennai – 600 026, India.

The cell lines were maintained in minimal essential medium (MEM) supplemented
with 10% FBS, 100 units/ml if penicillin and 100 µg/ml of streptomycin at 37ºC in a
humified incubator (5% CO2 and 95% air). The cell line was grown as a monolayer in a
humified atmosphere at 37ºC with CO2 in 25 cm2 falcon flasks. The cell growth was
found to be exponential, after 2-3 days of seeding.

The experiments were performed with the cells in the logarithmic phase of growth and
were removed by trypsinisation and harvested with 0.15% trypsin and 0.08%
EDTA, then harvested twice with PBS.
Experimental set up for in vitro studies

Negative Control Vero cell line
Group I-
Control Vero cells
Group II- Vero cells treated with Adriamycin
Group III- Vero cells treated with Adriamycin + Quercetin
Group IV- Vero cells treated with Quercetin

Anticancer and hepatoprotective effect of Quercetin on HepG2 cell line
Group I-
Control HepG2 cells
Group II-
HepG2 cells treated with Adriamycin
Group III- HepG2 cells treated with Adriamycin + Quercetin
Group IV- HepG2 cells treated with Quercetin
RESULTS
Table 1 showing the cell viability of HepG2 and vero cells on treatment with
adriamycin, quercetin and combination by MTT assay
S.
No.
Conc
(µg/ml)
HepG2 cell
treated with ADR
HepG2 cell
treated with
QUER
HepG2 cell
treated with ADR
+ QUER
Vero cells treated
with ADR
Vero cells treated
with QUER
1
100
20.75 ± 1.09
18.85±1.32
23.75 ± 1.17
12.25±0.95
42.95 ±1.22
2
50
34.58 ± 1.25
32.45 ±0.95
36.78 ± 0.87
18.36 ±0.36
58.98 ±0.63
3
25
39.61 ± 1.08
45.65 ±0.85
42.38 ± 1.45
22.56 ±0.45
63.72 ±0.75
4
12.5
49.68 ± 0.63
58.32 ±0.45
55.75 ± 1.03
34.58 ±1.02
78.89 ±1.02
5
6.25
59.74 ± 0.62
67.95 ±1.35
63.38 ± 0.68
54.45 ±1.35
86.23 ±0.65
6
3.125
71.69±1.08
78.95 ±1.49
7
1.56
79.24 ± 1.09
91.15 ±0.385
73.89 ± 1.55
85.56 ± 0.64
8
Cell
control
100
100
100
68.89 ±0.85
90.25 ±0.98
85.56 ±0.96
95.18 ± 1.23
100
100
Cell viability %
Fig 1. Effect of adriamycin and quercetin on IC50 value on HepG2 cell line
100
90
80
70
60
50
40
30
20
10
0
adr
quer
adr + quer
100
50
25
12.5
6.25
3.125
1.56
Concentration µg / ml
Fig 2. Effect of adriamycin, and quercetin on IC50 value on vero cell line
Cell viability %
100
80
60
adr
40
quer
20
0
100
50
25
12.5
6.25
Concentration µg / ml
3.125
1.56
EFFECT OF ADRIAMYCIN AND QUERCETIN ON LIPID PEROXIDATION AND ANTIOXIDANT ENZYMES
LPO nmoles of MDA / mg
protein
Fig 3. Effect of adriamycin and quercetin on lipid peroxidation in HepG2 and vero cell lines
40
35
30
25
20
15
10
5
0
HepG2
a NS
a*
a*b*
a*b@
a*b*c#
VERO
a # b*c*
GROUP I
GROUP II
GROUP III
GROUP IV
catalase (µmoles of
H2O2 consumed/min/mg
protein
Fig 4.Effect of adriamycin and quercetin on catalase activity in HepG2 and vero cell lines
2.5
a*b*c*
a*b*
2
a NSb*
1.5
a*b*c @
a NS
a*
1
0.5
0
GROUP I
GROUP II
GROUP III
Values are ± SD, n = 3
a – as compared with group I, b - as compared with group II, c - as compared with group III
Statistical significance * p < 0.001, @ p < 0.01, # p < 0.05
GROUP IV
HepG2
VERO
SOD activity (U/mg
protein)
Fig 5.Effect of adriamycin and quercetin on the activity of SOD in HepG2 and vero cell lines
16
14
12
10
8
6
4
2
0
a*b*
a*b*c**
a NS
HepG2
a*b*
a# b*c*
VERO
a#
GROUP I
GROUP II
GROUP III
GROUP IV
GR (nmoles of NADPH
oxidized/min/mg protein)
Fig 6. Effect of adriamycin and quercetin on glutathione reductase activity in HepG2 and vero cell lines
1.4
a*b*
HepG2
VERO
1.2
a*b*c*
1
a@b*c*
aNSb*
a@
a*
0.8
0.6
0.4
0.2
0
GROUP I
GROUP II
GROUP III
Values are ± SD, n = 3
a – as compared with group I, b - as compared with group II, c - as compared with group III
Statistical significance * p < 0.001, @ p < 0.01, # p < 0.05
GROUP IV
Fig 7. Effect of adriamycin and quercetin on glutathione peroxidase activity in HepG2
and vero cell lines
Gpx activity (µmoles/min/mg
protein)
1.4
a*b*
HepG2
1.2
a@b#
1
a*b*c*
aNSb*
aNS
a*
0.8
0.6
0.4
0.2
0
GROUP I
GROUP II
GROUP III
GROUP IV
Values are ± SD, n = 3
a – as compared with group I, b - as compared with group II, c - as compared with group III
Statistical significance * p < 0.001, @ p < 0.01, # p < 0.05
VERO
EFFECT OF ADRIAMYCIN AND QUERCETIN ON MARKER ENZYMES
Fig 8. Effect of adriamycin and quercetin on alkaline phosphatase activity in HepG2 and vero cell lines
Activity of ALP in µmoles of
phenol liberated/min/mg
protein
14
a*
HepG2
a@
12
a* b@ c#
a*
10
a* b*
VERO
a# b@
8
a@ b* c*
6
4
2
0
GROUP I
GROUP II
GROUP III
GROUP IV
Fig 9. Effect of adriamycin and quercetin on lactate dehydrogenase activity in HepG2
a*
and vero
cell lines
a*
Activity of LDH in µmoles
of pyruvate
liberated/min/mg protein
8
7
a#
aNS bNS
a* bNS
6
HepG2
aNS bNS cNS
VERO
b*c *
5
4
3
2
1
0
GROUP I
GROUP II
GROUP III
Values are ± SD, n = 3
a – as compared with group I, b - as compared with group II, c - as compared with group III
Statistical significance * p < 0.001, @ p < 0.01, # p < 0.05
GROUP IV
Fig 10. mRNA Expressions of Bcl-xl, Bcl 2, p21, p53, caspase 9, caspase 3 in adriamycin and quercetin
1
2
treated HepG2 cells
3
4
5
1 – Marker
2 – control HepG2 cells (Group I )
3 –HepG2 cells treated with adriamycin (Group II)
4 –HepG2 cells treated with adriamycin and quercetin (Group III)
5 –HepG2 cells treated with quercetin(Group IV)
Fig 11. Protein Expression of Bcl 2, Bak, Apaf, Bax, p 53, p21, caspase 3, caspase 9 and PARP in adriamycin
1
and quercetin treated HepG2 cells
2
3
4
1 – control HepG2 cells treated (Group I )
2 –HepG2 cells treated with adriamycin (Group II)
3 –HepG2 cells treated with adriamycin and quercetin (Group III)
4 –HepG2 cells treated with quercetin(Group IV)
SUMMARY
The present study was done to evaluate the hepatoprotective activity of quercetin along with
adriamycin and the following parameters were analysed.

The Cell viability of the cell lines were assayed by MTT Assay, quercetin was found to
protect the normal cells as well enhance the adriamycin action.

The enzymic antioxidant activities of Superoxide dismutase, catalase, glutathione reductase,
Glutathione peroxidise, alkaline phosphatase, lactate dehydrogenase and levels of lipid
peroxidation were analysed and was proved to protect the cell lines by its enhancing antioxidant
power.

DNA fragmentation was analysed by Agarose Gel electrophoresis in HepG2 cell lines and
quercetin was found to be equally responsible in causing cell death through apoptosis.

mRNA expression of Bcl-xl, Bcl 2, p21, p53, caspase 9, caspase 3 in adriamycin and
quercetin treated HepG2 cells was done and the apoptosis was confirmed by the antioxidant
power of quercetin.

Protein expression of Bcl 2, Bak, Apaf, Bax, p 53, p21, caspase 3, caspase 9 and
PARP was analysed using Western Blot in HepG2 and H9C2 cell lines also proved the
efficacy of quercetin to enhance apoptosis.
Adriamycin, an anthracycline antibiotic, is widely used in the treatment of a variety of
human malignancies, including liver cancer, breast cancer, small cell carcinoma of the lung
and acute leukemia's (Blum and Carter, 1974). Like most of the anticancer drugs, adriamycin
also causes various toxic effects, the commonest of which is the dose-dependent
cardiotoxicity which leads to acute and chronic heart failure (Koima et al., 1999).
The present study has also proved that quercetin, a flavonoid antioxidant is a promising
anticancer agent by itself and when it was used in combination with adriamycin, it was able to
enhance the anticancer effect of adriamycin as well as protect the normal cells.
Relevance to the society

The present study was aimed in creating an awareness among common people
about the importance of comsuming foods rich in flavonoids and antioxidants which
may protect us from diseases like cancer or in future can be used as a supplementation
to prevent side effects in cancer patients who are treated with chemotherapeutic
drugs.