DrR VENKATESWARI MSc PhD DEPARTMENT OF MEDICAL BIOCHEMISTRY Dr ALM PG INSTITUTE OF BASIC MEDICAL SCIENCES UNIVERSITY OF MADRAS TARAMANI CHENNAI 600 113 INDIA 6 th WORLD CONGRESS ON BIOTECHNOLOGY ID: 749187
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Slide1
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 SCIENCESUNIVERSITY OF MADRASTARAMANI, CHENNAI – 600 113, INDIA
6
th
WORLD CONGRESS ON BIOTECHNOLOGYSlide2
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).Slide3
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. Slide4
HEPATITIS
B VIRUS
HEPATITIS C VIRUS
ALCOHOL
LIVER CANCERCAUSATIVE AGENTS OF LIVER CANCER
AFLATOXINSlide5
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 high-risk behaviors such as intravenous drug use and alcohol abuse. Diagnosis of Liver CancerThere 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. Slide6
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).Slide7
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. Slide8
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. Slide9
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. Slide10
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 of the hydroxyl (-OH) substitutions [Hardwood M, 2007]. Structure of QuercetinQUERCETINSlide11
VEGETABLES
APPLES
GRAPES
ONIONS
BERRIESGREEN TEASlide12
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).Slide13
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.Slide14
IN VITRO STUDIESCell 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.Slide15
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). Slide16
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% CO
2 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.Slide17
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
QuercetinSlide18
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
73.89 ± 1.55
68.89 ±0.85
90.25 ±0.98
7
1.56
79.24 ± 1.0991.15 ±0.38585.56 ± 0.6485.56 ±0.96 95.18 ± 1.23 8Cell control100100100100100
Table 1 showing the cell viability of HepG2 and vero cells on treatment with adriamycin, quercetin and combination by MTT assay
RESULTSSlide19
Fig 2. Effect of adriamycin, and quercetin
on IC50 value on vero cell line
Fig 1. Effect of
adriamycin
and quercetin on IC50 value on HepG2 cell lineSlide20
Fig 3. Effect of adriamycin and
quercetin on lipid peroxidation in HepG2 and
vero cell lines
Fig 4.Effect of adriamycin
and quercetin on catalase activity in HepG2 and vero cell linesValues are ± SD, n = 3a – as compared with group I, b - as compared with group II, c - as compared with group IIIStatistical significance * p < 0.001, @ p < 0.01, # p < 0.05
a NS
a*b*
a*b*c#
a NS
a*b*c @
a*
a*b@
a # b*c*
a
NSb
*
EFFECT OF ADRIAMYCIN AND QUERCETIN ON LIPID PEROXIDATION AND ANTIOXIDANT ENZYMESSlide21
Fig 5.Effect of
adriamycin and
quercetin on the activity of SOD in HepG2 and vero cell lines
Fig 6. Effect of
adriamycin and quercetin on glutathione reductase activity in HepG2 and vero cell linesValues are ± SD, n = 3a – as compared with group I, b - as compared with group II, c - as compared with group IIIStatistical significance * p < 0.001, @ p < 0.01, # p < 0.05
a NS
a*b*
a*b*c**
a#
a# b*c*
a@
a*b*
a*b*c*
aNSb
*
a@b
*c*
a*Slide22
Fig 7. Effect of
adriamycin and
quercetin on glutathione peroxidase activity in HepG2 and vero cell lines
Values are ± SD, n = 3a – 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 < 0.05aNSa*b*a@b#aNSb
*
a*b*c*
a*Slide23
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 < 0.05Fig 8. Effect of adriamycin
and quercetin
on alkaline phosphatase activity in HepG2 and vero cell linesFig 9. Effect of adriamycin and quercetin on lactate dehydrogenase activity in HepG2 and vero cell lines
a@
a* b*
a* b@ c#
a*
a# b@
a@ b* c*
a*
a#
a
NS
b
NS
a
NS
b
NS
c
NS
a*
a*
bNS
b*c *
a*
EFFECT OF ADRIAMYCIN AND QUERCETIN ON MARKER ENZYMESSlide24
Fig 10. mRNA Expressions of
Bcl-xl,
Bcl 2, p21, p53, caspase 9, caspase 3 in
adriamycin and
quercetin treated HepG2 cells12345
1 – Marker2 – 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)Slide25
Fig 11. Protein Expression of
Bcl 2,
Bak, Apaf, Bax, p 53, p21, caspase
3,
caspase 9 and PARP in adriamycin and quercetin treated HepG2 cells1234
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)Slide26
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
.
SUMMARYSlide27
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
.Slide28
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.Slide29
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