quercetin enhances the effect of adriamycin in human hepatocellular carcinoma (hepg2) cell lines...

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 MADRAS

TARAMANI, CHENNAI – 600 113, INDIA

6th WORLD CONGRESS ON BIOTECHNOLOGY

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.

HEPATITIS B VIRUS

HEPATITIS C VIRUS

ALCOHOL

LIVER CANCER

CAUSATIVE AGENTS OF 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 high-

risk 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.

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 Quercetin

QUERC

ETIN

VEGETABLES

APPLES GRAPES

ONIONSBERRIES 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

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 ±1.4973.89 ± 1.55

68.89 ±0.85 90.25 ±0.98

7 1.56 79.24 ± 1.09 91.15 ±0.385 85.56 ± 0.64 85.56 ±0.96 95.18 ± 1.23

8 Cell control

100 100 100 100 100

Table 1 showing the cell viability of HepG2 and vero cells on treatment with adriamycin, quercetin and combination by MTT assay

RESULTS

Fig 2. Effect of adriamycin, and quercetin on IC50 value on vero cell line

100 50 25 12.5 6.25 3.125 1.560

20

40

60

80

100

adrquer

Concentration µg / ml

Cell v

iabilit

y %

100 50 25 12.5 6.25 3.125 1.560

102030405060708090

100

adr

quer

adr + quer

Concentration µg / ml

Cel

l via

bilit

y %

Fig 1. Effect of adriamycin and quercetin on IC50 value on HepG2 cell line

Fig 3. Effect of adriamycin and quercetin on lipid peroxidation in HepG2 and vero cell lines

GROUP I GROUP II GROUP III GROUP IV0

0.5

1

1.5

2

2.5HepG2

VERO

cata

lase

(µ

mol

es o

f H

2O2

cons

umed

/min

/mg

prot

ein a*b*

a*

a*b*c*

Fig 4.Effect of adriamycin and quercetin on catalase activity in HepG2 and vero cell lines

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


5

10

15

20

25

30

35

40HepG2

VERO

LP

O n

mol

es o

f M

DA

/ m

g pr

otei

na 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 ENZYMES


10121416

HepG2

VERO

SO

D a

ctiv

ity (

U/m

g pr

o-te

in)

a*b*

Fig 5.Effect of adriamycin and quercetin on the activity of SOD in HepG2 and vero cell lines


0.2

0.4

0.6

0.8

1

1.2

1.4 HepG2

VERO

GR

(nm

oles

of N

AD

PH

oxid

ized

/min

/mg

prot

ein)

Fig 6. Effect of adriamycin and quercetin on glutathione reductase activity in HepG2 and vero cell lines




a NS

a*b*

a*b*c**

a#a# b*c*

a@

a*b*

a*b*c*

aNSb*a@b*c*

a*


0.2

0.4

0.6

0.8

1

1.2

1.4HepG2

VERO

Gpx

act

ivity

(µ

mol

es/m

in/m

g pr

o-te

in)

Fig 7. Effect of adriamycin and quercetin on glutathione peroxidase activity in HepG2

and vero cell lines




aNS

a*b*

a@b#

aNSb*a*b*c*

a*


2

4

6

8

10

12

14

HepG2

VERO

Act

ivit

y of

AL

P in µ

mole

s of

phenol libera

ted/

min

/mg p

rote

in


1

2

3

4

5

6

7HepG2

VERO

Act

ivit

y of

LD

H in µ

mole

s of

pyr

uva

te lib

era

ted/

min

/mg p

rote

in




Fig 8. Effect of adriamycin and quercetin on alkaline phosphatase activity in HepG2 and vero cell lines

Fig 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# aNS bNSaNS bNS cNS

a*a* bNS

b*c *

a*

EFFECT OF ADRIAMYCIN AND QUERCETIN ON MARKER ENZYMES

Fig 10. mRNA Expressions of Bcl-xl, Bcl 2, p21, p53, caspase 9, caspase 3 in adriamycin and quercetin

treated HepG2 cells1 2 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

and quercetin treated HepG2 cells1 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)

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.

SUMMARY

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.

THANK YOU

quercetin enhances the effect of adriamycin in human hepatocellular carcinoma (hepg2) cell lines...

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