Hepatoprotective Effect of Chenopodium Album On Ccl4-Induced Liver Damage and Metabolites act as Dual Topoisomerase Inhibitors and Induce Apoptosis In The Mcf7 Cell Line In Rats ABSTRACT Liver is one of the vital organs involved in various responsibilities required to maintain homeostasis of our body

Hepatoprotective Effect of Chenopodium Album On Ccl4-Induced
Liver Damage and Metabolites act as Dual
Topoisomerase Inhibitors and Induce Apoptosis In
The Mcf7 Cell Line
In Rats
ABSTRACT
Liver is one of the vital organs involved in various responsibilities required to maintain homeostasis of our body. In addition, liver is involved in removal of toxins and drugs from the circulatory system. Hence, the concern for hepatotoxcity is in rise throughout the World. Conventional drugs used in the hepatotoxic management are mostly inadequate and have serious adverse effects. In spite of the tremendous strides in modern medicine, there are grossly few drugs that stimulate liver function, offer protection to the liver from damage or help regeneration of hepatic cells. Hence, the present study aims in synthesis of novel Schiff base compound using S-allycystiene and methionine. The newly synthesized compound, 2-2-(2-Allylthio)-Carboxyethylimino) Ethylideneamino)-4-(Methylthio) Butanoic Acid (ACEMB) was physically characterized using FTIR, 1HNMR, GC-MS, UV and TLC. ACEMB showed potent in vitro antioxidant property. The protective effect of ACEMB against the well accepted hepatotoxicant (CCl4) model of oxidative stress-induced liver injury in rat was elucidated. ACEMB attenuated the leakage of markers of liver injury, such as, enzymes (AST, ALT, GGT, ALP and LDH), biomolecules (bilirubin, total bilirubin, conjugated bilirubin) in to the blood circulation. Also, ACEMB preserved the protein synthesis function of liver, which was evidenced by normalization of the concentration of total proteins, albumin and globulin to control level in the plasma compared to CCl4-induced toxic rat. In addition, ACEMB protected against the CCl4-induced kidney toxicity by reducing secretion of creatinine, urea and uric acid in the serum, which was enhanced by CCl4. These protective effects of ACEMB was due to its antioxidant property, which was revealed by reduced oxidative stress (TBARS and HP) and enhanced functions of endogenous antioxidative system (SOD, catalase, GPx, GST, GSH, vitamin E and C)in comparison to CCl4 intoxicated rat alone. In addition, ACEMB protected the mitochondrial functional activities thereby would have maintained the ATP content of hepatocyte and averted the apoptosis/necrosis in the hepatocytes. ACEMB protected the liver against dyslipidemia during CCl4 intoxication. The biochemical alterations are in concurrence with the histological observations, wherein ACEMB pretreatment normalized the vacuolated hepatocytes, necrosis and degenerated nuclei present in the CCl4-induced toxic liver. In silico analysis pointed out the interaction of ACEMB with cytochrome P450 and COX-2. However, detailed molecular analysis is required to ascertain the role of ACEMB in the function of cytochrome P450 and COX-2.

The protective activity of ACEMB in the present study is comparable to the standard drug silymarin. Chronic administration of ACEMB prior to CCl4 toxicity ameliorated the damage to the liver. It is speculated that ACEMB mediates hepatoprotective effect by substituting itself as an antioxidant, increasing the activity of the endogenous antioxidant system, preserving the structure of hepatic parenchyma, decreasing the dyslipidemia, preserving the ER and improving the mitochondrial function against CCl4 toxicity in rat.

Desgalactotigonin (DGT) and oleanolic acid 3-O-?-D-glucuronide (OAG) were isolated from Chenopodium album seeds, characterized using spectral analysis and evaluated for cytotoxic activity against various cancer cell lines. DGT and OAG induced apoptosis in a human breast cancer cell line (MCF-7) and were found to effectively inhibit human topoisomerases I and II in vitro. The study of the mode of enzyme inhibition revealed that both acted as catalytic inhibitors. IC50 values for DGT and OAG on MCF-7 cells were found to be 8.27 ?M and 11.33 ?M respectively. Using FACS and western blot analysis, the compounds were observed to block the cell cycle at S phase. Activation of caspases 7 and 9 suggested that caspase pathways were involved in inducing apoptosis.

Materials and Methods
Plant material
Matured fruits of Chenopodium album (Linn) were collected from the medicinal plant garden of kollimalai . The plant is not an endangered or protected species. It was authenticated by Dr. Debjani Basu, Asst. Director, Botanical Survey of India, Howrah (West Bengal, India). A voucher specimen (No.786) was deposited in the Chemistry Department, Indian Institute of Chemical Biology. The black seeds were segregated from the pericarp of the fruits and then ground in an industrial blender.

Preparation of extract
Powdered seeds (3 Kg) were defatted by successive percolation with petroleum ether (60-80°C) and chloroform, then extracted by percolation with methanol. The solvent was distilled off under reduced pressure (<50 °C) using a rotary evaporator (Eyela, Tokyo, Japan). Removal of inorganic salts from the methanol extract was effected by partitioning between n-butanol and water. The n-butanol part was concentrated by reduced pressure distillation to yield 35g of greenish brown mass
Purification of extract
The greenish brown mass obtained from the n-butanolic portion was passed through silica gel (60-120) column and eluted with solvents in an increasing order of polarity starting with chloroform and ending with methanol. The fractions were further purified through repeated column chromatography in conjunction with thin layer chromatography. This ultimately yielded two pure products, the major one with eluent CHCl3: MeOH (75:25) and the minor one with eluent CHCl3: MeOH (65:35); the rest were inseparable mixtures. The major product (yield 0.004% on dry weight basis of the plant material), which was crystallized from MeOH and characterized as desgalactotigonin via spectroscopic analysis, viz. mass, 13C NMR, and 1H NMR spectroscopy followed by comparison with data reported in the literature (Yan et al., 1996). This is the first report of its isolation from Chenopodium album. The other product was characterized as oleanolic acid 3- O-?-D-glucoronide (OAG; yield 0.0009%) (Lavaud et al., 2000)
Cell culture, chemicals
MCF-7 cells (Michigan Cancer Foundation-7) were cultured in Dulbecco’s Modified Eagles Medium (DMEM) (GIBCO, Invitrogen, Carlsbad, CA, US) supplemented with 10% fetal bovine serum (GIBCO), 1X PSN (GIBCO) and gentamycin (GIBCO). Cells were incubated in a humidified CO2 incubator at 37°C. Camptothecin (CPT) and etoposide (ETO) were purchased from Sigma (St. Louis, MO, US) and dissolved in dimethylsulphoxide (DMSO).

Cell viability assay
Cells were seeded in 96 well plates. After 24 h, these were treated with respective compounds keeping the DMSO concentration less than 0.5%. After 72 h of treatment, cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide (MTT) assay. Briefly cells were washed with 1X PBS and treated with MTT for 4 h at 37°C. The precipitates were dissolved in DMSO and plates were analyzed on Thermo MULTISKAN EX plate reader at 595 nm
DNA relaxation assay for topoisomerase I and II enzymes
Recombinant human DNA topoisomerase I and II enzymes were purchased from TopoGEN Inc (Port Orange, Florida, USA). DNA relaxation assay for human topoisomerase I was performed in the presence or absence of respective compounds by briefly incubating 100 fmol of supercoiled pBS SK(+) DNA with 50 fmol of the enzyme in a reaction buffer containing 25 mM Tris-Cl (pH 7.5), 5% glycerol, 50 mM KCl, 0.5 mM DTT, 10 mM MgCl2, 25 mM EDTA and 150 ?g/ml bovine serum albumin, as described previously (Ray S., et al 1998). DNA relaxation assay for human topoisomerase II was performed in the presence and absence of respective compounds by briefly
incubating 100 fmol of supercoiled pBS SK(+) DNA with 50 fmol of the enzyme in a reaction buffer containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, 0.5 mM DTT and 30 ?g/ml bovine serum albumin (the reactions buffers were provided with the enzyme by the manufacturer). The reactions were incubated at 37 °C for 30 minutes, loaded on 1% agarose gel and subjected to electrophoresis at 20 volts overnight. After completion of electrophoresis, gels were stained with 0.5 ?g/ml ethidium bromide and viewed by Gel Doc 2000 (BioRad) under
UV illumination. DNA Relaxation was assessed by monitoring the decreased electrophoretic mobility of relaxed topoisomerase of pBS SK(+) DNA. Camptothecin (CPT) and etoposide (ETO), were used as a positive control inhibitors for topoisomerase I and II, respectively.

DNA cleavage assays for topoisomerase I and II enzymes
DNA cleavage assays for topoisomerase I and II were performed in the presence or absence of respective compounds by briefly incubating 100 fmol of supercoiled pBS SK(+) DNA with 500 fmol of topo I or topo II enzyme. For topoisomerase I, the reaction buffer contained 10 mM Tris-Cl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA and 15 ?g/ml bovine serum albumin. For topoisomerase II, the reaction buffer contained 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, 0.5 mM DTT and 30 ?g/ml bovine serum albumin. The reactions were incubated at 37 °C for 30 minutes and stopped with 0.5% SDS. Enzymes were digested by proteinase
K treatment. Reactions were loaded on 1% agarose gel containing 0.5 ?g/ml ethidium bromide. The gel was subjected to electrophoresis at 80 volts for 3 hours. After completion of electrophoresis, gels were viewed by Gel Doc 2000 (BioRad) under UV illumination. Camptothecin (CPT) and etoposide (ETO) were used as positive control inhibitors that stabilize topoisomerase I-DNA and topoisomerase II–DNA covalent complexes, respectively.

Immunoband depletion assay
For immunoband depletion assay MOLT-4 cells were cultured in 35 mm dishes separately in the presence of 10 ?M each of CPT, ETO, DGT, and OAG, and harvested at different time points. For pretreatment immunoband depletion assay, cells were first treated with either 10 ?M DGT or 10 ?M OAG for 2 h and then treated with 10 ?M CPT or 10 ?M ETO. Equal amounts of protein were electrophoresed on SDS-poly acryl amide gel, separated proteins were transferred on to nitrocellulose membrane, and western blotting was performed using anti-topo I and anti-topo II
antibodies (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA).

Apoptosis assay
Briefly, MCF-7 cells (1×106/ml) were incubated with or without DGT (IC50: 8.27 ?M) and OAG (IC50: 11.33 ?M) for 24 h and 48 h at 37°C, 5% CO2. Cells were then washed twice in PBS and resuspended in Annexin-V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2; pH 7.4). Annexin-V-FITC was then added according to the manufacturer’s instructions (Calbiochem, USA) and incubated for 15 min under dark conditions at 25°C. PI (0.1 ?g/ml) was added just prior to acquisition. Data were acquired using BD LSR Fortessa flowcytometer (Becton Dickinson, USA) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm, and analyzed with
BD FACS Diva software (Becton Dickinson, USA).

Detection of mitochondrial membrane potential
The mitochondria are attractive targets for cancer chemotherapy since its impairment renders cells non viable. The loss of mitochondrial potential is one of the indicators of apoptosis. The mitochondrial transmembrane electrochemical gradient (??m) was measured using mitochondrial potential sensor JC-1, a cell permeable, cationic and lipophilic dye. In viable cells, it freely crosses the mitochondrial membrane and forms J-aggregates which fluoresce red. In apoptotic cells, decrease in mitochondrial membrane potential prevents JC-1 from entering the
mitochondria and remains as monomers in the cytosol that emits a predominantly green fluorescence (Deeb, D., et all., 2010). Therefore, the ratio of Jaggregates/monomers functions as an effective indicator of mitochondrial transmembrane potential and helps distinguish apoptotic cells from their healthy counterparts. Briefly, MCF-7 cells (1×106/ml) were incubated with IC50 concentrations of DGT and OAG for 24 h and 48 h at 37°C, 5% CO2. The cells were then washed with PBS, and incubated with JC-1 (2 ?M) according to manufacturer’s protocol (Molecular Probes, USA) under dark conditions for 15-30 min at 37°C, 5% CO2. Cells were acquired using FACS and analyzed
using FACS Diva software. CCCP was used as positive control. Probes, USA) under dark conditions for 15-30 min at 37°C, 5% CO2. Cells were acquired using FACS and analyzedusing FACS Diva software. CCCP was used as positive control.

Measurement of ROS generation
The effect of IC50 concentration of DGT and OAG on generation of ROS (12 and 24 h) was measured in cellsn (1×106/ml). After treatment, cells were washed with PBS and resuspended in PBS, and then incubated with H2DCFDA (20 ?M in PBS) for 30 min at 37°C. Subsequently, cells were again washed and resuspended in PBS. DCF fluorescence was determined by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 530 nm by BD LSR Fortessa flowcytometer.

Western blot analysis of caspases and PARP-1 cleavage
Cells were cultured in 6 cm dishes and treated with either 8.27 ?M DGT or 11.33 ?M OAG for 48 h. Cells were lysed in NP-40 buffer and equal amounts of proteins were electrophoresed on SDS-polyacrylamide gel. Separated proteins were then transferred on to nitrocellulose membrane and detected using antibodies available in Apoptosis Antibody Sampler Kit (Cell Signaling Technology) by western blotting.

Cell cycle analysis using flow cytometry
Sub confluent cells were treated with IC50 concentration of DGT or OAG in culture medium as described above for 24 h and 48 h. The cells were then harvested, washed with cold PBS, and processed for cell cycle analysis. Briefly, 1×106 cells were resuspended in 300 ?l of cold PBS to which 70% cold ethanol (700 ?l) was added, and the cells were then incubated overnight at 4°C. After removing ethanol and washing with PBS, cells were suspended in 500 ?l PBS, and incubated with 100 ?g/ml RNase A for 1 h at 37°C. The cells were subsequently incubated with 50 ?g/ml propidium iodide (PI) for another 30 min at 37°C in subdued light (Carpinelli et al., 2011). The stained cell suspension was analyzed with BD LSR Fortessa flowcytometer. The DNA content of 10,000 cells per sample was used to analyze the cell cycle using DNA histograms. The DNA content in the cell-cycle of the analyzed cells was calculated by MODFIT 3.0 software (Verity Software House, ME, USA).

Assessment of cell morphology
Cells (3×104/well) were grown in 6-well TC plates and treated with or without DGT and OAG at IC50 concentration for 24 h. Morphological changes were observed with an inverted phase contrast microscope (Model: OLYMPUS IX70, Olympus Optical Co. Ltd., Shibuya-ku, Tokyo, Japan) and photographs were taken with the help of a digital camera (Olympus, Inc. Japan). See Fig-7 in manuscript
Introduction
Breast cancer, a common malignancy in women, is a condition that starts in the cells of the breast, travels through lymphatic vessels, begins to grow in lymph nodes and can metastasize or spread throughout the body. The number of new cases of breast cancer has jumped dramatically worldwide from about 640 000 in 1980 to more than 1.6 million in
2010.1 Chemotherapy is predominantly used for the treatment of breast cancer in stages 2–4 and combination drugs are usually administered for periods of 3–6 months. Currently
anthracyclines, e.g. doxorubicin and daunorubicin (topoisomerase II inhibitors), are widely used for the treatment of breast cancer. One of the most common regimens used is ‘AC’, a combination of Adriamycin (doxorubicin) and Cyclophosphamide. However, these agents show serious side effects and in many cases cause cardiotoxicity. These chemotherapeutic
drugs destroy rapidly growing cancer cells either by causing DNA damage upon replication or by other mechanisms.

They also damage fast-growing normal cells which leads to serious side effects and, in particular, heart muscle cell damage which is one of the most dangerous complications
of doxorubicin treatment.2–5 Drugs discovered from different natural sources, especially
from plants, have hugely benefited the human race. A recent report from the WHO states that about 80% of the world’s population relies on traditional medicine.6 In the last three
decades more than a hundred anticancer agents have been developed of which nine are pure natural products, twenty five are natural product derivatives, eighteen are natural
product mimics and eleven candidates are derived from a natural product pharmacophore.7 Our group has made concerted efforts in identifying bioactive natural products for different diseases from Indian medicinal plants and as a part of that endeavour we have dealt with Chenopodium album, a fast growing weedy annual plant used as a vegetable and cattle feed, for its bioactivity and bioactive constituents.8–10 Reports show that Chenopodium album leaf extracts exhibit several pharmacological activities such as antibacterial,11 hepatoprotective,12 and anticancer.13We preferred to analyze the seeds due partly to their abundance as each plant produces tens of thousands of black seeds.

Apoptosis is programmed cell death that plays essential roles in the development and homeostasis of multi-cellular organisms. 15 Thus, we designed a set of experiments that entail detailed apoptotic events induced by these compounds, e.g. the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, flipping of
phosphatidylserine in plasma membranes, cleavage of caspase-9, caspase-7 and PARP-1, etc., along with a focus on topoisomerase inhibitory activities, as DNA topoisomerases are important cellular enzymes and are targets of several anticancer and antibacterial drugs. The findings of the study are reported in this communication.

Conclusion
In conclusion, our results demonstrate that the compounds DGT and OAG exhibit potent anticancer activities on breast cancer cells by inhibiting both DNA topoisomerase I and II and inducing apoptotic cell death. The availability of the plant material and the simplicity of the isolation procedure also demonstrate that DGT and OAG have bright prospects for further exploitation as anti-breast cancer agents.

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