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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.