Search results for: HepG2
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 53

Search results for: HepG2

53 The Role of Il-6-Mediated NS5ATP9 Expression in Autophagy of Liver Cancer Cells

Authors: Hongping Lu, Kelbinur Tursun, Yaru Li, Yu Zhang, Shunai Liu, Ming Han

Abstract:

Objective: To investigate whether NS5ATP9 is involved in IL-6 mediated autophagy and the relationship between IL-6 and NS5ATP9 in liver cancer cells. Methods: 1. Detect the mRNA and protein levels of Beclin 1 after HepG2 cells were treated with or without recombinant human IL-6 protein. 2. Measure and compare of the changes of autophagy-related genes with their respective control, after IL-6 was silenced or neutralized with monoclonal antibody against human IL-6. 3. HepG2 cells were incubated with 50 ng/ml of IL-6 in the presence or absence of PDTC. The expression of NS5ATP9 was analyzed by Western blot after 48 h. 4. After NS5ATP9-silenced HepG2 cells had been treated with 50 ng/ml recombinant IL-6 protein, we detected the Beclin 1 and LC3B (LC3Ⅱ/Ⅰ) expression. 5. HepG2 cells were transfected with pNS5ATP9, si-NS5ATP9, and their respective control. Total RNA was isolated from cells and analyzed for IL-6. 6. Silence or neutralization of IL-6 in HepG2 cells which has been transfected with NS5ATP9. Beclin 1 and LC3 protein levels were analyzed by Western blot. Result: 1. After HepG2 were treated with recombinant human IL-6 protein, the expression of endogenous Beclin 1 was up-regulated at mRNA and protein level, and the conversion of endogenous LC3-I to LC3-II was also increased. These results indicated that IL-6 could induce autophagy. 2. When HepG2 cells were treated with IL-6 siRNA or monoclonal antibody against human IL-6, the expression of autophagy-related genes were decreased. 3. Exogenous human IL-6 recombinant protein up-regulated NS5ATP9 via NF-κB activation. 4. The expression of Beclin 1 and LC3B was down-regulated after IL-6 treated NS5ATP9-silenced HepG2 cells. 5. NS5ATP9 could reverse regulates IL-6 expression in HepG2 cells. 6. Silence or neutralization of IL-6 attenuates NS5ATP9-induced autophagy slightly. Conclusion: Our results implied that in HCC patients, maybe the higher level of IL-6 in the serum promoted the expression of NS5ATP9 and induced autophagy in cancer cells. And the over-expression of NS5ATP9 which induced by IL-6, in turn, increased IL-6 expression, further, promotes the IL-6/NS5ATP9-mediated autophagy and affects the progression of tumor. Therefore, NS5ATP9 silence might be a potential target for HCC therapy.

Keywords: autophagy, Hepatocellular carcinoma, IL-6, microenvironment, NS5ATP9

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52 Hexane Extract of Thymus serpyllum L.: GC-MS Profile, Antioxidant Potential and Anticancer Impact on HepG2 (Liver Carcinoma) Cell Line

Authors: Salma Baig, Bakrudeen Ali Ahmad, Ainnul Hamidah Syahadah Azizan, Hapipah Mohd Ali, Elham Rouhollahi, Mahmood Ameen Abdulla

Abstract:

Free radical damage induced by reactive oxygen species (ROS) contributes to etiology of many chronic diseases, cancer being one of them. Recent studies have been successful in ROS targeted therapies via antioxidants using mouse models in cancer therapeutics. The present study was designed to scrutinize anticancer activity, antioxidant activity of 5 different extracts of Thymus serpyllum in MDA-MB-231, MCF-7, HepG2, HCT-116, PC3, and A549. Identification of the phytochemicals present in the most active extract of Thymus serpyllum was conducted using gas chromatography coupled with mass spectrophotometry and antioxidant activity was measured by using DPPH radical scavenging and FRAP assay. Anticancer impact of the extract in terms of IC50 was evaluated using MTT cell viability assay. Results revealed that the hexane extract showed the best anticancer activity in HepG2 (Liver Carcinoma Cell Line) with an IC50 value of 23 ± 0.14 µg/ml followed by 25 µg/ml in HCT-116 (Colon Cancer Cell Line), 30 µm/ml in MCF-7 (Breast Cancer Cell Line), 35 µg/ml in MDA-MB-231 (Breast Cancer Cell Line), 57 µg/ml in PC3 (Prostate Cancer Cell Line) and 60 µg/ml in A549 (Lung Carcinoma Cell Line). GC-MS profile of the hexane extract showed the presence of 31 compounds with carvacrol, thymol and thymoquione being the major compounds. Phenolics such as Vitamin E, terpinen-4-ol, borneol and phytol were also identified. Hence, here we present the first report on cytotoxicity of hexane extract of Thymus serpyllum extract in HepG2 cell line with a robust anticancer activity with an IC50 of 23 ± 0.14 µg/ml.

Keywords: Thymus serpyllum L., hexane extract, GC-MS profile, antioxidant activity, anticancer activity, HepG2 cell line

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51 Initiation of Paraptosis-Like PCD Pathway in Hepatocellular Carcinoma Cell Line by Hep88 mAb through the Binding of Mortalin (HSPA9) and Alpha-Enolase

Authors: Panadda Rojpibulstit, Suthathip Kittisenachai, Songchan Puthong, Sirikul Manochantr, Pornpen Gamnarai, Sasichai Kangsadalampai, Sittiruk Roytrakul

Abstract:

Hepatocellular carcinoma (HCC) is the most primary hepatic cancer worldwide. Nowadays a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is continually developed in HCC treatment. In this regard, after establishing and consequently exploring Hep88 mAb’s tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb’s specific Ag from both membrane and cytoplasmic fractions of HepG2 cell line was identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E.coli BL21 (DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1-3 days was investigated using a transmission electron microscope. The result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) andalpha-enolase. In addition, gradual appearance of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Taken together, paraptosis-like programmed cell death (PCD) of HepG2 is induced by binding of mortalin (HSPA9) and alpha-enolase to Hep88 mAb. Mortalin depletion by formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independent of p53-mediated apoptosis. Additionally, Hep88 mAb-alpha-enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction. These results imply that Hep88 mAb might be a promising tool for development of an effective treatment of HCC in the next decade.

Keywords: Hepatocellular carcinoma, Monoclonal antibody, Paraptosis-like program cell death, Transmission electron microscopy, mortalin (HSPA9), alpha-enolase

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50 The Effect of Thymoquinone and Sorafenib Combination on Hepatocellular Carcinoma Cell Line

Authors: Nabila N. El-Maraghy, Amany Essa, Yousra Abdel–Mottaleb, Nada Ismail

Abstract:

The use of combination of chemotherapy and natural products to influence the cell death with low doses of chemotherapeutic agents and few side effects has recently emerged as a new method of cancer therapy. Aim: Evaluation the modulatory effect of Thymoquinone on HepG2 cells treated with Sorafenib. Methods: Hepatocellular Carcinoma HepG2 cell line was treated with Sorafenib and TQ individually and in combination. The effect of these treatments on cell viability (MTT assay), apoptosis (Expression of Caspase-3) and oxidative markers (GSH content and extent of lipid peroxidation) was determined. Results: When compared the effect of both agents alone and the combination of the IC50 of Sorafenib and the IC50 TQ, the combination resulted in reduction of cell inhibition and apoptosis and antagonize their actions on GSH content and extent of lipid peroxidation which are increased. This study showed potent anti-tumor activity of both TQ and Sorafenib separately on HepG2 but upon combination surprisingly they interacted and give antagonistic effect. Conclusion: Co-treatment resulted in antagonistic interaction between Sorafenib and Thymoquinone.

Keywords: antagonism, hepatocellular carcinoma, sorafenib, thymoquinone

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49 Investigation of Cytotoxic Compounds in Ethyl Acetate and Chloroform Extracts of Nigella sativa Seeds by Sulforhodamine-B Assay-Guided Fractionation

Authors: Harshani Uggallage, Kapila D. Dissanayaka

Abstract:

A Sulforhodamine-B assay-guided fractionation on Nigella sativa seeds was conducted to determine the presence of cytotoxic compounds against human hepatoma (HepG2) cells. Initially, a freeze-dried sample of Nigella sativa seeds was sequentially extracted into solvents of increasing polarities. Crude extracts from the sequential extraction of Nigella sativa seeds in chloroform and ethyl acetate showed the highest cytotoxicity. The combined mixture of these two extracts was subjected to bioassay guided fractionation using a modified Kupchan method of partitioning, followed by Sephadex® LH-20 chromatography. This chromatographic separation process resulted in a column fraction with a convincing IC50 (half-maximal inhibitory concentration) value of 13.07µg/ml, which is considerable for developing therapeutic drug leads against human hepatoma. Reversed phase High-Performance Liquid Chromatography (HPLC) was finally conducted for the same column fraction, and the result indicates the presence of one or several main cytotoxic compounds against human HepG2 cells.

Keywords: cytotoxic compounds, half-maximal inhibitory concentration, high-performance liquid chromatography, human HepG2 cells, nigella sativa seeds, Sulforhodamine-B assay

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48 Cratoxy Formosum (Jack) Dyer Leaf Extract-Induced Human Breast and Liver Cancer Cells Death

Authors: Benjaporn Buranrat, Nootchanat Mairuae

Abstract:

Cratoxylum formosum (Jack) Dyer (CF) has been used for the traditional medicines in South East Asian and Thailand. Normally, northeast Thai vegetables have proven cytotoxic to many cancer cells. Therefore, the present study aims to explore the molecular mechanisms underlying CF-induced cancer cell death and apoptosis on breast and liver cancer cells. The cytotoxicity and antiproliferative effects of CF on the human breast MCF-7 and liver HepG2 cancer cell lines were evaluated using sulforhodamine B assay and colony formation assay. Cell migration assay was measured using wound healing assay. The apoptosis induction mechanisms were investigated through reactive oxygen species formation, caspase 3 activity, and JC-1 activity. Gene expression by real-time PCR and apoptosis related protein levels by Western blot analysis. CF induced MCF-7 and HepG2 cell death by time- and dose-dependent manner. Furthermore, CF had the greater cytotoxic potency on MCF-7 more than HepG2 cells with IC50 values of 85.70+4.52 μM and 219.03±9.96 μM respectively, at 24 h. Treatment with CF also caused a dose-dependent decrease in colony forming ability and cell migration, especially on MCF-7 cells. CF induced ROS formation, increased caspase 3 activities, and decreased the mitochondrial membrane potential, and causing apoptotic body production and DNA fragmentation. CF significantly decreased expression of the cell cycle regulatory protein RAC1 and downstream proteins, cdk6. Additionally, CF enhanced p21 and reduced cyclin D1 protein levels. CF leaf extract induced cell death, apoptosis, antimigration in both of MCF-7 and HepG2 cells. CF could be useful for developing to anticancer drug candidate for breast and liver cancer therapy.

Keywords: cratoxylum formosum (jack) dyer, breast cancer, liver cancer, cell death

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47 Thermodynamic and Immunochemical Studies of Antibody Biofunctionalized Gold Nanoparticles Mediated Photothermal Ablation in Human Liver Cancer Cells

Authors: Lucian Mocan, Flaviu Tabaran, Teodora Mocan, Cristian Matea, Cornel Iancu

Abstract:

We present method of Gold Nanoparticle enhanced laser thermal ablation of HepG2 cells (Human hepatocellular liver carcinoma cell line), based on a simple gold nanoparticle carrier system, such as serum albumin (BSA), and demonstrate its selective therapeutic efficacy. Hyperspectral, contrast phase, and confocal microscopy combined immunochemical staining were used to demonstrate the selective internalization of HSA-GNPs via Gp60 receptors and the caveolin-mediated endocytosis inside HepG2 cells. We examined the ability of laser-activated carbon nanotubes to induce Hsp70 expression using confocal microscopy. Hep G2 cells heat-shocked (laser activated BSA-GNPs) to 42°C demonstrated an up-regulation of Hsp70 compared with control cells (BSA-GNPs treated cells without laser), which showed no detectable constitutive expression of Hsp70. We observed a time-dependent induction in Hsp70 expression in Hep G2 treated with BSA-GNPs and LASER irradiated. The post-irradiation apoptotic rate of HepG2 cells treated with HSA-GNPs ranged from 88.24% (for 50 mg/L) at 60 seconds, while at 30 minute the rate increased to 92.34% (50 mg/L). These unique results may represent a major step in liver cancer treatment using nanolocalized thermal ablation by laser heating.

Keywords: gold nanoparticles, liver cancer, albumin, laser irradiation

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46 Gas Chromatography-Analysis, Antioxidant, Anti-Inflammatory, and Anticancer Activities of Some Extracts and Fractions of Linum usitatissimum

Authors: Eman Abdullah Morsi, Hend Okasha, Heba Abdel Hady, Mortada El-Sayed, Mohamed Abbas Shemis

Abstract:

Context: Linum usitatissimum (Linn), known as Flaxseed, is one of the most important medicinal plants traditionally used for various health as nutritional purposes. Objective: Estimation of total phenolic and flavonoid contents as well as evaluate the antioxidant using α, α-diphenyl-β-picrylhydrazyl (DPPH), 2-2'azinobis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and total antioxidant capacity (TAC) assay and investigation of anti-inflammatory by Bovine serum albumin (BSA) and anticancer activities of hepatocellular carcinoma cell line (HepG2) and breast cancer cell line (MCF7) have been applied on hexane, ethyl acetate, n-butanol and methanol extracts and also, fractions of methonal extract (hexane, ethyl acetate and n-butanol). Materials and Methods: Phenolic and flavonoid contents were detected using spectrophotometric and colorimetric assays. Antioxidant and anti-inflammatory activities were estimated in-vitro. Anticancer activity of extracts and fractions of methanolic extract were tested on (HepG2) and (MCF7). Results: Methanolic extract and its ethyl acetate fraction contain higher contents of total phenols and flavonoids. In addition, methanolic extract had higher antioxidant activity. Butanolic and ethyl acetate fractions yielded higher percent of inhibition of protein denaturation. Meanwhile, ethyl acetate fraction and methanolic extract had anticancer activity against HepG2 and MCF7 (IC50=60 ± 0.24 and 29.4 ± 0.12µg.mL⁻¹) and (IC50=94.7 ± 0.21 and 227 ± 0.48µg.mL⁻¹), respectively. In Gas chromatography-mass spectrometry (GC-MS) analysis, methanolic extract has 32 compounds, whereas; ethyl acetate and butanol fractions contain 40 and 36 compounds, respectively. Conclusion: Flaxseed contains totally different biologically active compounds that have been found to possess good variable activities, which can protect human body against several diseases.

Keywords: phenolic content, flavonoid content, HepG2, MCF7, hemolysis-assay, flaxseed

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45 Protective Potential of Hyperhalophilic Diatoms Extract Against Lead Induced Oxidative Stress in Rats and Human HepG2 and HEK293 Cells Line

Authors: Wassim Guermazi, Saoussan Boukhris, Neila Annabi Trabelsi, Tarek Rebai, Alya Sellami-Kamoun, Habib Ayadi

Abstract:

This work investigates the protective effects of the microalga Halamphora sp. extract (H. Ext) as a natural product on lead-intoxicated liver and kidney human cells in vitro and in vivo on rats wistar. HepG2 cells line derived from human hepatocellular carcinoma and HEK293 cells line derived from human embryonic kidney were used for the in vitro study. The analysis of the fatty acids methyl esters of the extract was performed by a GC/MS. Four groups of rats, each of which was composed of six animals, were used for the in vivo experiment. The pretreatment of HepG2 and HEK293 cells line with the extract (100 µg mL-1) significantly (p < 0.05) protected against cytotoxicity induced by lead exposure. In vivo, the biochemical parameters in serum, namely malondialdehyde level (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, were measured in supernatants of organ homogenates. H. Ext was found to be rich in fatty acids, essentially palmitic and palmitoleic accounting respectively 29.46% and 42.07% of total fatty acids. Both in vitro and in vivo, the co-treatment with H. Ext allowed the protection of the liver and kidney cells structure, as well as the significant preservation of normal antioxidant and biochemical parameters in rats. Halamphora extract rich in fatty acids has been proven to be effective in protection against Pb-induced toxicity.

Keywords: microalga extract, human cells line, fatty acid, lead exposure, oxidative stress, rats

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44 Design and Facile Synthesis of New Amino Acid Derivatives with Anti-Tumor and Antimicrobial Activities

Authors: Hoda Sabry Othman, Randa Helmy Swellem, Galal Abd El-Moein Nawwar

Abstract:

N-cyanoacetyl glycine is a reactive polyfunctional precursor for synthesis of new difficult accessible compounds including pyridones, thiazolopyridine and others. The key step of this protocol is the formation of different ylidines which underwent Michael addition with carbon nucleophiles affording various heterocyclic compounds. Selected compounds underwent pharmacological evaluation, in vitro against two cell lines; breast cell line (MCF-7),and liver cell line(HEPG2). Compounds 14, 15a and 16 showed IC50 values 8.93, 8.18 and 8.03 (µ/ml) respectively for breast cell line (MCF-7), while the standard drug (Tamoxifen) revealed IC50 8.31. With respect to the liver cell line (HEPG2), compounds 14 and 15a revealed IC50 18.4 and 13.6(µ/ml) respectively while the IC50 of the standard drug(5-Flurouracil) is 25(µ/ml). The antimicrobial activity was also screened and revealed that oxime 7 and ylidine 9f showed a broad-spectrum activity.

Keywords: antitumor, cyanoacetyl glycine, heterocycles, pyridones

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43 Nanotechnology-Based Treatment of Liver Cancer

Authors: Lucian Mocan

Abstract:

We present method of Nanoparticle enhanced laser thermal ablation of HepG2 cells (Human hepatocellular liver carcinomacell line), using gold nanoparticles combuned with a specific growth factor and demonstrate its selective therapeutic efficacy usig ex vivo specimens. Ex vivo-perfused liver specimens were obtained from hepatocellular carcinoma patients similarly to the surgical technique of transplantation. Ab bound to GNPs was inoculated intra-arterially onto the resulting specimen and determined the specific delivery of the nano-bioconjugate into the malignant tissue by means of the capillary bed. The extent of necrosis was considerable following laser therapy and at the same time surrounding parenchyma was not seriously affected. The selective photothermal ablation of the malignant liver tissue was obtained after the selective accumulation of Ab bound to GNPs into tumor cells following ex-vivo intravascular perfusion. These unique results may represent a major step in liver cancer treatment using nanolocalized thermal ablation by laser heating.

Keywords: HepG2 cells, gold nanoparticles, nanoparticle functionalization, laser irradiation

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42 Cytotoxic Activity against Hepatocarcinoma and Cholangiocarcinoma Cells of Four Cathartic Herbal Medicines

Authors: Pranporn Kuropakornpong, Srisopa Ruangnoo, Arunporn Itharat

Abstract:

Liver cancer has the highest prevalence rate in the North and Northeast of Thailand. Four Thai medicinal plants such as resin of Ferula asafoetida Regel, latex of Aloe barbadensis Miller leaves, roots of Baliospermum manotanum, and latex of Garcinia hanburyi Hook are used in Thai traditional medicine as cathartic drug and detoxification in liver cancer patients. Thus, this research aimed to evaluate the cytotoxic activity of these plants against hepatocarcinoma (HepG2) and cholangiocarcinoma (KKU-M156) cells by SRB assay. These plants were macerated in 95% ethanol. The results showed that roots of Baliospermum manotanum and latex of Garcinia hanburyi Hook showed the strongest cytotoxicity against HepG2 (IC50 = 3.03+0.91 and 0.62+0.01µg/ml, respectively) and KKU-M156 (IC50 = 0.978+0.663 and 0.006+0.005 µg/ml, respectively). Latex of Garcinia hanburyi Hook also showed high cytotoxicity against normal cell line (IC50=8.86+0.31 µg/ml), and even though its selective values are high, dose of this herb should be limited.

Keywords: cholangiocarcinoma, cytotoxic activity, Garcinia hanburyi Hook, hepatocarcinoma

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41 Cytotoxic Metabolites from Tagetes minuta L. Growing in Saudi Arabia

Authors: Ali A. A. Alqarni, Gamal A. Mohamed, Hossam M. Abdallah, Sabrin R. M. Ibrahim

Abstract:

Phytochemical investigation of the methanolic extract of aerial parts of Tagetes minuta L. (Family: Asteraceae) using different chromatographic techniques led to the isolation of five compounds; ecliptal (1), scopoletin (2), P-hydroxy benzoic acid (3), patuletin (4), and patuletin-7-O-β-D-glucopyranoside (5) (Figure 1). Their structures were established based on physical, chemical, and spectral data [Ultraviolet (UV), Proton ¹H, Carbon thirteen ¹³C, and Heteronuclear Multiple Bond Correlation (HMBC) NMR], as well as Electrospray Ionization Mass Spectroscopy (ESIMS) and comparison with literature data. Their cytotoxic activity was assessed towards human liver hepatocellular carcinoma (HepG2), human breast cancer (MCF-7), and human colon cancer (HCT116) cancer cell lines using sulphorhodamine B (SRB) assay. It is noteworthy that compound 1 demonstrated a significant cytotoxic potential towards HepG2, MCF7, and HCT116 cells with IC₅₀s ranging from 2.74 to 7.01 μM, compared to doxorubicin (IC₅₀ 0.18, 0.60, and 0.20 μM, respectively), whereas compounds 2, 4, and 5 showed moderate cytotoxic potential with IC50s ranging from 11.71 to 35.64 μM. However, 3 was inactive up to a concentration of 100 μM towards the three tested cancer cell lines.

Keywords: Asteraceae, cytotoxicity, metabolites, Tagetes minuta

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40 Glucose Uptake Rate of Insulin-Resistant Human Liver Carcinoma Cells (IR/HepG2) by Flavonoids from Enicostema littorale via IR/IRS1/AKT Pathway

Authors: Priyanka Mokashi, Aparna Khanna, Nancy Pandita

Abstract:

Diabetes mellitus is a chronic metabolic disorder which will be the 7th leading cause of death by 2030. The current line of treatment for the diabetes mellitus is oral antidiabetic drugs (biguanides, sulfonylureas, meglitinides, thiazolidinediones and alpha-glycosidase inhibitors) and insulin therapy depending upon the type 1 or type 2 diabetes mellitus. But, these treatments have their disadvantages, ranging from the developing of resistance to the drugs and adverse effects caused by them. Alternative to these synthetic agents, natural products provides a new insight for the development of more efficient and safe drugs due to their therapeutic values. Enicostema littorale blume (A. Raynal) is a traditional Indian plant belongs to the Gentianaceae family. It is widely distributed in Asia, Africa, and South America. There are few reports on Swrtiamarin, major component of this plant for its antidiabetic activity. However, the antidiabetic activity of flavonoids from E. littorale and their mechanism of action have not yet been elucidated. Flavonoids have a positive relationship with disease prevention and can act on various molecular targets and regulate different signaling pathways in pancreatic β-cells, adipocytes, hepatocytes and skeletal myofibers. They may exert beneficial effects in diabetes by (i) improving hyperglycemia through regulation of glucose metabolism in hepatocytes; (ii) enhancing insulin secretion and reducing apoptosis and promoting proliferation of pancreatic β-cells; (iii) increasing glucose uptake in hepatocytes, skeletal muscle and white adipose tissue (iv) reducing insulin resistance, inflammation and oxidative stress. Therefore, we have isolated four flavonoid rich fractions, Fraction A (FA), Fraction B (FB), Fraction C (FC), Fraction D (FD) from crude alcoholic hot (AH) extract from E. littorale, identified by LC/MS. Total eight flavonoids were identified on the basis of fragmentation pattern. Flavonoid FA showed the presence of swertisin, isovitexin, and saponarin; FB showed genkwanin, quercetin, isovitexin, FC showed apigenin, swertisin, quercetin, 5-O-glucosylswertisin and 5-O-glucosylisoswertisin whereas FD showed the presence of swertisin. Further, these fractions were assessed for their antidiabetic activity on stimulating glucose uptake in insulin-resistant HepG2 cell line model (IR/HepG2). The results showed that FD containing C-glycoside Swertisin has significantly increased the glucose uptake rate of IR/HepG2 cells at the concentration of 10 µg/ml as compared to positive control Metformin (0.5mM) which was determined by glucose oxidase- peroxidase method. It has been reported that enhancement of glucose uptake of cells occurs due the translocation of Glut4 vesicles to cell membrane through IR/IRS1/AKT pathway. Therefore, we have studied expressions of three genes IRS1, AKT and Glut4 by real-time PCR to evaluate whether they follow the same pathway or not. It was seen that the glucose uptake rate has increased in FD treated IR/HepG2 cells due to the activation of insulin receptor substrate-1 (IRS1) followed by protein kinase B (AKT) through phosphoinositide 3-kinase (PI3K) leading to translocation of Glut 4 vesicles to cell membrane, thereby enhancing glucose uptake and insulin sensitivity of insulin resistant HepG2 cells. Hence, the up-regulation indicated the mechanism of action through which FD (Swertisin) acts as antidiabetic candidate in the treatment of type 2 diabetes mellitus.

Keywords: E. littorale, glucose transporter, glucose uptake rate, insulin resistance

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39 Biochemical Effects of Low Dose Dimethyl Sulfoxide on HepG2 Liver Cancer Cell Line

Authors: Esra Sengul, R. G. Aktas, M. E. Sitar, H. Isan

Abstract:

Hepatocellular carcinoma (HCC) is a hepatocellular tumor commonly found on the surface of the chronic liver. HepG2 is the most commonly used cell type in HCC studies. The main proteins remaining in the blood serum after separation of plasma fibrinogen are albumin and globulin. The fact that the albumin showed hepatocellular damage and reflect the synthesis capacity of the liver was the main reason for our use. Alpha-Fetoprotein (AFP) is an albumin-like structural embryonic globulin found in the embryonic cortex, cord blood, and fetal liver. It has been used as a marker in the follow-up of tumor growth in various malign tumors and in the efficacy of surgical-medical treatments, so it is a good protein to look at with albumins. We have seen the morphological changes of dimethyl sulfoxide (DMSO) on HepG2 and decided to investigate its biochemical effects. We examined the effects of DMSO, which is used in cell cultures, on albumin, AFP and total protein at low doses. Material Method: Cell Culture: Medium was prepared in cell culture using Dulbecco's Modified Eagle Media (DMEM), Fetal Bovine Serum Dulbecco's (FBS), Phosphate Buffered Saline and trypsin maintained at -20 ° C. Fixation of Cells: HepG2 cells, which have been appropriately developed at the end of the first week, were fixed with acetone. We stored our cells in PBS at + 4 ° C until the fixation was completed. Area Calculation: The areas of the cells are calculated in the ImageJ (IJ). Microscope examination: The examination was performed with a Zeiss Inverted Microscope. Daytime photographs were taken at 40x, 100x 200x and 400x. Biochemical Tests: Protein (Total): Serum sample was analyzed by a spectrophotometric method in autoanalyzer. Albumin: Serum sample was analyzed by a spectrophotometric method in autoanalyzer. Alpha-fetoprotein: Serum sample was analyzed by ECLIA method. Results: When liver cancer cells were cultured in medium with 1% DMSO for 4 weeks, a significant difference was observed when compared with the control group. As a result, we have seen that DMSO can be used as an important agent in the treatment of liver cancer. Cell areas were reduced in the DMSO group compared to the control group and the confluency ratio increased. The ability to form spheroids was also significantly higher in the DMSO group. Alpha-fetoprotein was lower than the values of an ordinary liver cancer patient and the total protein amount increased to the reference range of the normal individual. Because the albumin sample was below the specimen value, the numerical results could not be obtained on biochemical examinations. We interpret all these results as making DMSO a caretaking aid. Since each one was not enough alone we used 3 parameters and the results were positive when we refer to the values of a normal healthy individual in parallel. We hope to extend the study further by adding new parameters and genetic analyzes, by increasing the number of samples, and by using DMSO as an adjunct agent in the treatment of liver cancer.

Keywords: hepatocellular carcinoma, HepG2, dimethyl sulfoxide, cell culture, ELISA

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38 Chemical Analysis and Cytotoxic Evaluation of Asphodelus Aestivus Brot. Flowers

Authors: Mai M. Farid, Mona El-Shabrawy, Sameh R. Hussein, Ahmed Elkhateeb, El-Said S. Abdel-Hameed, Mona M. Marzouk

Abstract:

Asphodelus aestivus Brot. Is a wild plant distributed in Egypt and is considered one of the five Asphodelus spp. from the family Asphodelaceae; it grows in dry grasslands and on rocky or sandy soil. The chemical components of A. aestivus flowers extract were analyzed using different chromatographic and spectral techniques and led to the isolation of two anthraquinones identified as emodin and emodin-O-glucoside. In addition to, five flavonoid compounds;kaempferol,Kaempferol-3-O-glucoside,Apigenin-6-C-glucoside-7-O-glucoside (Saponarine), luteolin 7-O-β-glucopyranoside, Isoorientin-O-malic acid which is a new compound in nature. The LC-ESI-MS/MS analysis of the flower extract of A. aestivus led to the identification of twenty- two compounds characterized by the presence of flavones, flavonols, and flavone C-glycosides. While GC/MS analysis led to the identification of 24 compounds comprising 98.32% of the oil, the major components of the oil were 9, 12, 15-Octadecatrieoic acid methyl ester 28.72%, and 9, 12-Octadecadieroic acid (Z, Z)-methyl ester 19.96%. In vitro cytotoxic activity of the aqueous methanol extract of A. aestivus flowers against HEPG2, HCT-116, MCF-7, and A549 culture was examined and showed moderate inhibition (62.3±1.1)% on HEPG2 cell line followed by (36.8±0.2)% inhibition on HCT-116 and a weak inhibition (5.7± 0.0.2) on MCF-7 cell line followed by (4.5± 0.4) % inhibition on A549 cell line and this is considered the first cytotoxic report of A. aestivus flowers.

Keywords: Anthraquinones, Asphodelus aestivus, Cytotoxic activity, Flavonoids, LC-ESI-MS/MS

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37 Berberine Ameliorates Glucocorticoid-Induced Hyperglycemia: An In-Vitro and In-Vivo Study

Authors: Mrinal Gupta, Mohammad Rumman, Babita Singh Abbas Ali Mahdi, Shivani Pandey

Abstract:

Introduction: Berberine (BBR), a bioactive compound isolated from Coptidis Rhizoma, possesses diverse pharmacological activities, including anti-bacterial, anti-inflammatory, antitumor, hypolipidemic, and anti-diabetic. However, its role as an anti-diabetic agent in animal models of dexamethasone (Dex)-induced diabetes remains unknown. Studies have shown that natural compounds, including aloe, caper, cinnamon, cocoa, green and black tea, and turmeric, can be used for treating Type 2 diabetes mellitus (DM). Compared to conventional drugs, natural compounds have fewer side effects and are easily available. Herein, we studied the anti-diabetic effects of BBR in a mice model of Dex-induced diabetes. Methods: HepG2 cell line was used for glucose release and glycogen synthesis studies. Cell proliferation was measured by methylthiotetrazole (MTT) assay. For animal studies, mice were treated with Dex (2 mg/kg, i.m.) for 30 days and the effect of BBR at the doses 100, 200, and 500 mg/kg (p.o.) was analyzed. Glucose, insulin, and pyruvate tests were performed to evaluate the development of the diabetic model. An echo MRI was performed to assess the fat mass. Further, to elucidate the mechanism of action of BBR, mRNA expression of genes regulating gluconeogenesis, glucose uptake, and glycolysis were analyzed. Results: In vitro BBR had no impact on cell viability up to a concentration of 50μM. Moreover, BBR suppressed the hepatic glucose release and improved glucose tolerance in HepG2 cells. In vivo, BBR improved glucose homeostasis in diabetic mice, as evidenced by enhanced glucose clearance, increased glycolysis, elevated glucose uptake, and decreased gluconeogenesis. Further, Dex treatment increased the total fat mass in mice, which was ameliorated by BBR treatment. Conclusion: BBR improves glucose tolerance by increasing glucose clearance, inhibiting hepatic glucose release, and decreasing obesity. Thus, BBR may become a potential therapeutic agent for treating glucocorticoid-induced diabetes and obesity in the future.

Keywords: glucocorticoid, hyperglycemia, berberine, HepG2 cells, insulin resistance, glucose

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36 Identification of Natural Liver X Receptor Agonists as the Treatments or Supplements for the Management of Alzheimer and Metabolic Diseases

Authors: Hsiang-Ru Lin

Abstract:

Cholesterol plays an essential role in the regulation of the progression of numerous important diseases including atherosclerosis and Alzheimer disease so the generation of suitable cholesterol-lowering reagents is urgent to develop. Liver X receptor (LXR) is a ligand-activated transcription factor whose natural ligands are cholesterols, oxysterols and glucose. Once being activated, LXR can transactivate the transcription action of various genes including CYP7A1, ABCA1, and SREBP1c, involved in the lipid metabolism, glucose metabolism and inflammatory pathway. Essentially, the upregulation of ABCA1 facilitates cholesterol efflux from the cells and attenuates the production of beta-amyloid (ABeta) 42 in brain so LXR is a promising target to develop the cholesterol-lowering reagents and preventative treatment of Alzheimer disease. Engelhardia roxburghiana is a deciduous tree growing in India, China, and Taiwan. However, its chemical composition is only reported to exhibit antitubercular and anti-inflammatory effects. In this study, four compounds, engelheptanoxides A, C, engelhardiol A, and B isolated from the root of Engelhardia roxburghiana were evaluated for their agonistic activity against LXR by the transient transfection reporter assays in the HepG2 cells. Furthermore, their interactive modes with LXR ligand binding pocket were generated by molecular modeling programs. By using the cell-based biological assays, engelheptanoxides A, C, engelhardiol A, and B showing no cytotoxic effect against the proliferation of HepG2 cells, exerted obvious LXR agonistic effects with similar activity as T0901317, a novel synthetic LXR agonist. Further modeling studies including docking and SAR (structure-activity relationship) showed that these compounds can locate in LXR ligand binding pocket in the similar manner as T0901317. Thus, LXR is one of nuclear receptors targeted by pharmaceutical industry for developing treatments of Alzheimer and atherosclerosis diseases. Importantly, the cell-based assays, together with molecular modeling studies suggesting a plausible binding mode, demonstrate that engelheptanoxides A, C, engelhardiol A, and B function as LXR agonists. This is the first report to demonstrate that the extract of Engelhardia roxburghiana contains LXR agonists. As such, these active components of Engelhardia roxburghiana or subsequent analogs may show important therapeutic effects through selective modulation of the LXR pathway.

Keywords: Liver X receptor (LXR), Engelhardia roxburghiana, CYP7A1, ABCA1, SREBP1c, HepG2 cells

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35 In Vitro Hepatoprotective and Anti-Hepatitis B Activitis of Cyperus rotundus Rhizome Fractions

Authors: Mohammad K. Parvez, Ahmed H. Arbab, Mohammed S. Al-Dosari

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Cyperus rotendus rhizomes are used as traditional medicine, including Ayurveda in chronic liver diseases and hepatitis B. We investigated the in vitro hepatoprotective and anti-hepatitis B virus (HBV) potential of Cyperus rotundus rhizome organic and aqueous fractions. Of these, the n-butanol and aqueous fractions showed the most promising, dose-dependent hepatoprotection in DCFH-injured HepG2 cells at 48 h. DCFH-toxicated cells were recovered to about 88% and 96%, upon treatment with n-butanol and aqueous fractions (200 g/ml), respectively compared to DCFH-only treated cells. Further, C. rotundus fractions were tested for anti-HBV activities by measuring the expression levels of viral antigens (HBsAg and HBeAg) in the HepG2.2.15 culture supernatants. At 48 h post-treatment, the ethyl acetate, n-butanol and aqueous fractions showed dose-dependent inhibition wherein at a higher dose (100 g/ml), HBsAg production was reduced to 60.27%, 46.87 and 42.76%, respectively. In a time-course study, HBsAg production was inhibited up to 50% and 40% by ethyl acetate and n-butanol fractions (100 g/ml), respectively on day 5. Three three active fractions were further subjected to time-dependent inhibition of HBeAg expression, an indirect measure of HBV active DNA replication. At day 5 post-treatment, ethyl acetate and n-butanol fractions downregulated HBV replication by 44.14% and 24.70%, respectively. In conclusion, our results showed very promising hepatoprotective and anti-HBV potential of C. rotendus tubers fractions in vitro. Our data could, therefore, provide the basis for the claimed traditional use of C. rotendus for jaundice and hepatitis.

Keywords: anti-hepatitis B, cyperus rotundus, hepatitis B virus, hepatoprotection

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34 Freshwater Cyanobacterial Bioactive Insights: Planktothricoides raciorskii Compounds vs. Green Synthesized Silver Nanoparticles: Characterization, in vitro Cytotoxicity, and Antibacterial Exploration

Authors: Sujatha Edla

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Introduction: New compounds and possible uses for the bioactive substances produced by freshwater cyanobacteria are constantly being discovered through research. Certain molecules are hazardous to the environment and human health, but others have potential applications in industry, biotechnology, and pharmaceuticals. These discoveries advance our knowledge of the varied functions these microbes perform in different ecosystems. Cyanobacterial silver nanoparticles (AgNPs) have special qualities and possible therapeutic advantages, which make them very promising for a range of medicinal uses. Aim: In our study; the attention was focused on the analysis and characterization of bioactive compounds extracted from freshwater cyanobacteria Planktothricoides raciorskii and its comparative study on Cyanobacteria-mediated silver nanoparticles synthesized by cell-free extract of Planktothricoides raciorskii. Material and Methods: A variety of bioactive secondary metabolites have been extracted, purified, and identified from cyanobacterial species using column chromatography, FTIR, and GC-MS/MS chromatography techniques and evaluated for antibacterial and cytotoxic studies, where the Cyanobacterial silver nanoparticles (CSNPs) were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) analysis and were further tested for antibacterial and cytotoxic efficiency. Results: The synthesis of CSNPs was confirmed through visible color change and shift of peaks at 430–445 nm by UV-Vis spectroscopy. The size of CSNPs was between 22 and 34 nm and oval-shaped which were confirmed by SEM and TEM analyses. The FTIR spectra showed a new peak at the range of 3,400–3,460 cm−1 compared to the control, confirming the reduction of silver nitrate. The antibacterial activity of both crude bioactive compound extract and CSNPs showed remarkable activity with Zone of inhibition against E. coli with 9.5mm and 10.2mm, 13mm and 14.5mm against S. paratyphi, 9.2mm and 9.8mm zone of inhibition against K. pneumonia by both crude extract and CSNPs, respectively. The cytotoxicity as evaluated by extracts of Planktothricoides raciorskii against MCF7-Human Breast Adenocarcinoma cell line and HepG2- Human Hepatocellular Carcinoma cell line employing MTT assay gave IC50 value of 47.18ug/ml, 110.81ug/ml against MCF7cell line and HepG2 cell line, respectively. The cytotoxic evaluation of Planktothricoides raciorskii CSNPs against the MCF7cell line was 43.37 ug/ml and 20.88 ug/ml against the HepG2 cell line. Our ongoing research in this field aims to uncover the full therapeutic potential of cyanobacterial silver nanoparticles and address any associated challenges.

Keywords: cyanobacteria, silvernanoparticles, pharmaceuticals, bioactive compounds, cytotoxic

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33 Screening of Potential Cytotoxic Activities of Some Medicinal Plants of Saudi Arabia

Authors: Syed Farooq Adil, Merajuddinkhan, Mujeeb Khan, Hamad Z. Alkhathlan

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Phytochemicals from plant extracts belong to an important source of natural products which have demonstrated excellent cytotoxic activities. However, plants of different origins exhibit diverse chemical compositions and bioactivities. Therefore, the discovery of plants based new anticancer agents from different parts of the world is always challenging. In this study, methanolic extracts of different parts of 11 plants from Saudi Arabia have been tested in vitro for their anticancer potential on human liver cancer cell line (HepG2). Particularly, for this study, plants from Asteraceae, Resedaceae, and Polygonaceae families were chosen on the basis of locally available ethnobotanical data and their medicinal properties. Among 12 tested extract samples, three samples obtained from Artemisia monosperma stem, Ochradenus baccatus aerial parts, and Pulicaria glutinosa stem have demonstrated interesting cytotoxic activities with a cell viability of 29.3%, 28.4% and 24.2%, respectively. Whereas, four plant extracts including Calendula arvensis aerial parts, Scorzonera musilii whole plant, A. monosperma leaves show moderate anticancer properties bearing a cell viability ranging from 11.9 to 16.7%. The remaining extracts have shown poor cytotoxic activities. Subsequently, GC-MS analysis of methanolic extracts of the four most active plants extracts such as C. comosum, O. baccatus, P. glutinosa and A. monosperma detected the presence of 41 phytomolecules. Among which 3-(4-hydroxyphenyl) propionitrile (1), 8,11-octadecadiynoic acid methyl ester (2), 6,7-dimethoxycoumarin (3), and 1-(2-hydroxyphenyl) ethenone (4) were found to be the lead compounds of C. comosum, O. baccatus P. glutinosa and A. monosperma, respectively.

Keywords: medicinal plants, asteraceae, polygonaceae, hepg2

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32 Toxicological Interactions of Silver Nanoparticles and Non-Essential Metals in Human Hepatocarcinoma Cell Line

Authors: Renata Rank Miranda, Arandi Ginane Bezerra, Ciro Alberto Oliveira Ribeiro, Marco AntôNio Ferreira Randi, Carmen Lúcia Voigt, Lilian Skytte, Kaare Lund Rasmussen, Francisco Filipak Neto, Frank Kjeldsen

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Synergetic and antagonistic effects of drugs are well-known concerns in pharmacological assessments of dose and toxicity. Similar approach should be used in assessing cellular uptake and cytotoxicity of nanoparticles. Since nanoparticles are released into the aquatic environment they may interact with existing xenobiotics. Here we used biochemical assays and quantitative proteomics to assess the cytotoxicity of silver nanoparticles (AgNP) when human hepatoma HepG2 cells were co-exposed to 2 nm AgNP together with either Cd2+ or Hg2+ ions. Time-course experiments (2h, 4h, and 24h) were conducted to assess the first response to the exposure studies. The general trend was that a synergetic toxicological response was observed in cells exposed to both AgNP and Cd2+ or Hg2+, with AgNP and Cd2+ being more toxic. This was observed by a significant increase in the ROS and superoxide level of >35% in the case of AgNP+Cd2+ compared to the sum of responses of AgNP and Cd2+, individually. Metabolic activity and viability also dropped more for AgNP+Cd2+ (>10%) than for AgNP and Cd2+ combined. We used inductively coupled plasma mass spectrometry to investigate if AgNP facilitates larger influx of toxic metal ions into HepG2 cells. Only Hg2+ ions was found to be more efficiently engulfed as the concentration of Hg2+ was found 2.8 times larger compared to exposure experiments with only Hg2+. This effect was not observed for Cd2+. We now continue with deep proteomics studies to obtain wider details on the mechanism of the toxicity related to AgNP, Cd2+, and AgNP+Cd2+, respectively.

Keywords: nanotoxicology, silver nanoparticles, proteomics, human cell line

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31 Antiplasmodial Activity of Drimane Sesquiterpene Isolated from Warburgia salutaris

Authors: Mthokozisi Simelane

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Background: Malaria remains a life-threatening disease in tropical regions despite the advances in the treatment of this disease, it still remains a significant burden as some parasites have become resistant to the currently available drugs. This has created a necessity for the development of alternative, more efficient antimalarial drugs. Warburgia salutaris is a traditional medicinal plant used in malaria treatment by Zulu traditional healers. Materials and methods: The W. salutaris stem-bark was extracted with dichloromethane and the compound was isolated through column chromatography. The compound was identified and characterized by spectroscopic analysis (1H NMR, 13C NMR, IR and MS) and the structure was also confirmed by x-ray crystallography. The anti-plasmodial activity (in vitro) was studied on NF54 Plasmodium falciparum strain (CQS). Cytotoxicity was measured using the MTT assay on HEK239 and HEPG2 cell lines. Docking of Mukaadial acetate was conducted in AutoDock Vina. Structural modifications were conducted in UCSF Chimera and molecular interactions examined in LigPlot. Results: The compound, Mukaadial Acetate showed appreciable inhibition (IC50 0.44±0.10 µg/ml) of the parasite growth and cytotoxicity activity of 0.124±0.109 and 0.199±0.083 (µg/ml) on HEK293 and HEPG2 cells respectively. Molecular docking revealed that Mukaadial Acetate binds to the purine, pyrophosphate and ribose binding sites of the PfHGXPRT with an optimum binding conformation and forms hydrogen bond, steric and hydrophobic interactions with the residues inhabiting the respective binding sites. Conclusion: It is apparent that W. salutaris contains components (including Mukaadial Acetate) that exhibit antimalarial activity. This study scientifically validates the use of this plant in folk medicine.

Keywords: plasmodium falciparum, molecular docking, antimalarial activity, PfHGXPRT, Warburgia salutaris, mukaadial acetate

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30 Antimutagenic Activity of a Protein, Lectin Fraction from Urtica Dioica L.

Authors: Nijole Savickiene, Antonella Di Sotto, Gabriela Mazzanti, Rasa Starselskyte, Silvia Di Giacomo, Annabella Vitalone

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Plant lectins are non-enzymic and non-immune origin proteins that specifically recognize and bind to various sugar structures and possess the activity to agglutinate cells and/or precipitate polysaccharides and glycoconjugates. The emerging evidences showed that plant lectins contribute not only to tumour cell recognition but also to cell adhesion and localization, to signal transduction, to mitogenic cytotoxicity and apoptosis. Among chitin-binding lectins, the Urtica dioica agglutinin (UDA), which is a complex of different isoforms, has been poorly studied for its biological activity. In this context and according to the increasing interest for lectins as novel antitumor drugs, present paper aimed at evaluating the potential antimutagenic activity of a lectin-like glycoprotein-enriched fraction from aerial part of Urtica dioica L. Aim: to evaluate the potential chemopreventive properties of a protein - lectin fraction from the aerial part of Urtica dioica. Materials and methods: Protein – lectin fraction has been tested for the antimutagenic activity in bacteria (50–800 mg/plate; Ames test by the preincubation method) and for the cytotoxicity on human hepatoma HepG2 cells (0.06–2 mg/mL; 24 and 48 h incubation). Results: Protein – lectin fraction from stinging nettle was not cytotoxic on HepG2 cells up to 2 mg/mL; conversely, it exhibited a strong antimutagenic activity against the mutagen 2-aminoanthracene (2AA) in all strains tested (maximum inhibition of 56.78 and 61% in TA98, TA100, and WP2uvrA strains, respectively, at 800 mg/plate). Discussion and conclusions: Protein – lectin fraction from Urtica dioica L. possesses antimutagenic and radical scavenging properties. Being 2AA a pro-carcinogenic agent, we hypothesize that the antimutagenicity of it can be due to the inhibition of CYP450-isoenzymes, involved in the mutagen bioactivation.

Keywords: lectins, antimutagenicity, chemoprevention, Urtica dioica

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29 A Replicon-Baculovirus Model for Efficient Packaging of Hepatitis E Virus RNA and Production of Infectious Virions

Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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28 Analysis of Cell Cycle Status in Radiation Non-Targeted Hepatoma Cells Using Flow Cytometry: Evidence of Dose Dependent Response

Authors: Sharmi Mukherjee, Anindita Chakraborty

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Cellular irradiation incites complex responses including arrest of cell cycle progression. This article accentuates the effects of radiation on cell cycle status of radiation non-targeted cells. Human Hepatoma HepG2 cells were exposed to increasing doses of γ radiations (1, 2, 4, 6 Gy) and their cell culture media was transferred to non-targeted HepG2 cells cultured in other Petri plates. These radiation non-targeted cells cultured in the ICCM (Irradiated cell conditioned media) were the bystander cells on which cell cycle analysis was performed using flow cytometry. An apparent decrease in the distribution of bystander cells at G0/G1 phase was observed with increased radiation doses upto 4 Gy representing a linear relationship. This was accompanied by a gradual increase in cellular distribution at G2/M phase. Interestingly the number of cells in G2/M phase at 1 and 2 Gy irradiation was not significantly different from each other. However, the percentage of G2 phase cells at 4 and 6 Gy doses were significantly higher than 2 Gy dose indicating the IC50 dose to be between 2 and 4 Gy. Cell cycle arrest is an indirect indicator of genotoxic damage in cells. In this study, bystander stress signals through the cell culture media of irradiated cells disseminated the radiation induced DNA damages in the non-targeted cells which resulted in arrest of the cell cycle progression at G2/M phase checkpoint. This implies that actual radiation biological effects represent a penumbra with effects encompassing a larger area than the actual beam. This article highlights the existence of genotoxic damages as bystander effects of γ rays in human Hepatoma cells by cell cycle analysis and opens up avenues for appraisal of bystander stress communications between tumor cells. Contemplation of underlying signaling mechanisms can be manipulated to maximize damaging effects of radiation with minimum dose and thus has therapeutic applications.

Keywords: bystander effect, cell cycle, genotoxic damage, hepatoma

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27 The Potential of Acanthaster Plancii Fractions as Anti-Atherosclerotic Agent by Inhibiting the Expression of Proprotein Convertase Subtilisin-Kexin Type 9

Authors: Nurjannatul Naim Kamaruddin, Tengku Sifziuzl Tengku Muhammad, Aina Farahiyah Abdul Manan, Habsah Mohamad

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Atherosclerosis which leads to cardiovascular diseases such as myocardial infarction, unstable angina (ischemic heart pain), sudden cardiac death and stroke is the principal cause of death worldwide. It has been a very critical issue as current common drug treatment, statin therapy has left bad side effects like rhabdomyolysis, atrial fibrillation, liver disease, abdominal and chest pain. Interestingly, the discoveries of proprotein convertase subtilisin-kexin type 9 have paved a new way in the treatment of atherosclerosis. This serine protease is believed to involve in the regulation of LDL- uptake by LDL-receptor. Therefore, this study was conducted to evaluate the potential of Acanthaster plancii fractions to reduce the transcriptional activity of the PCSK9 promoter. In this study, the marine organism which is Acanthaster plancii has been used as the source for marine compounds in inhibiting PCSK9. The cytotoxicity activity of ten fractions from the methanol extracts of Acanthaster plancii was investigated on HepG2 cell lines using MTS assay and dual glo luciferase assay was carried out later to analyses the effects of the samples in reducing the transcriptional activity of the PCSK9 promoter. Both assays used fractions with five different concentrations, 3.13µg/mL, 6.25µg/mL, 12.5µg/mL, 25µg/mL, and 50µg/mL. MTS assay indicated that the fractions are non-cytotoxic towards HepG2 cell lines as their IC50 value is greater than 30µg/mL. Whilst, for the dual glo luciferase assay, among all the fractions, Enhance Fraction 2 (EF2) showed the best potential in reducing the transcriptional activity of the PCSK9 promoter. The results indicated that this EF2 gave the lowest PCSK9 promoter expression at low concentration which is 0.2 fold change at 6.25µg/mL. This finding suggested that further analysis should be done to validate the potential of Acanthaster plancii as the source of anti-atherosclerotic agent.

Keywords: Acanthaster plancii, atherosclerosis, luciferase assay, PCSK9

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26 Comparative in vitro Anticancer Activity of Two Siddha Formulations: Neeradi Muthu Vallathymezugu and Thamira Kattu Chendooram

Authors: Vasudha Devi, Arul Amuthan, K. Narayanan, Praveen KS, Venkata Rao J

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Background: Siddha Medicine is one of the Indian traditional medical systems, in which the cancer disease is mentioned as 'putrunoi' which literally means the disease of growth like termite mound. There are number of formulations available for the treatment of cancer disease. Neeradi muthu vallathymezugu (NMV) and thamira kattu chendooram (TKC) are two drugs commonly prescribed by Siddha physicians. These drugs have been clinically reported to be safe and effective when given orally. Though these formulations are in practice for centuries, no efforts have been made to standardize them and explore their anti-cancer potential systematically. Objective: To compare the cytotoxic activity of NMV and TKC with doxorubicin using cancer cell lines. Materials and methods: For this study, ethanol extract of NMV was taken, whereas TKC was used as such. In vitro cytotoxic activity was evaluated by sulphorhodamine (SRB) assay against human hepatic cancer cells (HepG2), human breast cancer cells (MCF-7) and human cervical cancer cells [KeLa]. Doxorubicin was used as the standard. The SRB assay is based on the ability of cellular proteins to bind with sulphorhodamine-B. The number of live cells in drug treated cell lines directly affects the color formation in the assay, which is estimated calorimetrically by measuring the absorbance at 540 nm to calculate the cytotoxicity (inhibitory concentration - IC50 value) of the drug. Results: The IC50values of NMV, TKC and doxorubicin against HepG2 were 3.08 µg/ml, 20.21 µg/ml and 1.21µg/ml respectively. In MCF-7, it was 11.75 µg/ml, 17.67 µg/ml and 2.8µg/ml. In HeLa, the values were 24.76 µg/ml, 73.35 µg/ml and 1.12µg/ml. Conclusions: The study proves the possible anti-cancer potential of these two formulations. Compared to TKC, NMV showed good cytotoxic effect even at low dose. Human hepatic cancer cells responded well even at very low dose, when compared to other cancer cells. Though, cytotoxic potential of these compounds was found to be less compared to doxorubicin, the isolated lead compound may have the potential to be used as an anticancer drug clinically.

Keywords: Neeradi muthu vallathymezugu (Hydnocarpus laurifolia), thamira kattu chendooram, cytotoxicity, in-vitro, Siddha Medicine

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25 Hepatoprotective and Immunostimulative Properties of Medicinal Plants against Tuberculosis

Authors: Anna-Mari Kok, Carel B. Oosthuizen, Namrita Lall

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Tuberculosis (TB) is a disease caused by the bacterial pathogen Mycobacterium tuberculosis. It is associated with high mortality rates in both developing and developed countries. Many higher plants are found that are medicinally associated with tuberculosis infection. Plants belonging to thirteen families were selected, based on their traditional usage for tuberculosis and its associated symptoms. Eight plants showed the best antimycobacterial activities (MIC-value ≤ 500.0 µg/ml) against M. tuberculosis H37Rv. LS was found to have a minimum inhibitory concentration (MIC) of 125 µg/ml whereas, Tulbaghia violacea, Heteromorpha arborescens, Sutherlandia frutescens, Eucalyptus deglupta, and Plectranthus neochilus were found to have a MIC value of 250 µg/ml against M. tuberculosis H37Rv. Cytotoxicity values on U937 and HepG2 cells were obtained and the IC50 values ranged between 40 ±4.30 and > 400 µg/ml for the U937 cell line and 72.4 ±1.50 and > 400 µg/ml for the HepG2 cell line. Heteromorpha arborescens had the lowest IC50 value in both cell lines and therefore showed moderate levels of toxicity. Of the 19 samples that underwent the 2, 2- diphenyl- 1- picrylhydrazyl (DPPH) antioxidant assay, Eucalyptus deglupta and Melianthus major showed significant free radical scavenging activities with concentrations of 1.33 and 1.32 µg/ml respectively for the inhibition of DPPH. Hepatotoxicity induced by acetaminophen identified Searsia lancea with hepatoprotective activity of 59.37% at a ¼ IC50 concentration. Out of the 7 samples that were investigated for their immunomodulatory capabilities, Eucalyptus deglupta produced the most IL-12 with Sutherlandia frutescens also showing positive results for IL-12 production. In the present study, Eucalyptus deglupta showed the most promising results with good activity against M. tuberculosis with an MIC-value of 250 µg/ml. It also has potent antioxidant activity with an IC50 value of 1.33 µg/ml. This sample also stimulated high production of the cytokine, IL-12. Searsia lancea showed moderate antimycobacterial acticvity with an MIC-value of 500 µg/ml. The antioxidant potential also showed promising results with an IC50 value of 4.50 µg/ml. The hepatoprotective capability of Searsia lancea was 59.34% at a ¼ IC50 concentration. Another sample Sutherlandia frutescens showed effective antimycobacterial activity with an MIC-value of 250 µg/ml. It also stimulated production of IL-12 with 13.43 pg/ml produced. These three samples can be considered for further studies for the consideration as adjuvants for current tuberculosis treatment.

Keywords: adjuvant, hepatoprotection, immunomodulation, tuberculosis

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24 Caffeic Acid in Cosmetic Formulations: An Innovative Assessment

Authors: Caroline M. Spagnol, Vera L. B. Isaac, Marcos A. Corrêa, Hérida R. N. Salgado

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Phenolic compounds are abundant in the Brazilian plant kingdom and they are part of a large and complex group of organic substances. Cinnamic acids are part of this group of organic compounds, and caffeic acid (CA) is one of its representatives. Antioxidants are compounds which act as free radical scavengers and, in other cases, such as metal chelators, both in the initiation stage and the propagation of oxidative process. The tyrosinase, polyphenol oxidase, is an enzyme that acts at various stages of melanin biosynthesis within the melanocytes and is considered a key molecule in this process. Some phenolic compounds exhibit inhibitory effects on melanogenesis by inhibiting the tyrosinase enzymatic activity and therefore has been the subject of studies. However, few studies have reported the effectiveness of these products and their safety. Objectives: To assess the inhibitory activity of tyrosinase, the antioxidant activity of CA and its cytotoxic potential. The method to evaluate the inhibitory activity of tyrosinase aims to assess the reduction transformation of L-dopa into dopaquinone reactions catalyzed by the enzyme. For evaluating the antioxidant activity was used the analytical methodology of DPPH radical inhibition. The cytotoxicity evaluation was carried out using the MTT method (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), a colorimetric assay which determines the amount of insoluble violet crystals formed by the reduction of MTT in the mitochondria of living cells. Based on the results obtained during the study, CA has low activity as a depigmenting agent. However, it is a more potent antioxidant than ascorbic acid (AA), since a lower amount of CA is sufficient to inhibit 50% of DPPH radical. The results are promising since CA concentration that promoted 50% toxicity in HepG2 cells (IC50=781.8 μg/mL) is approximately 330 to 400 times greater than the concentration required to inhibit 50% of DPPH (IC50 DPPH= 2.39 μg/mL) and ABTS (IC50 ABTS= 1.96 μg/mL) radicals scavenging activity, respectively. The maximum concentration of caffeic acid tested (1140 mg /mL) did not reach 50% of cell death in HaCat cells. Thus, it was concluded that the caffeic acid does not cause toxicity in HepG2 and HaCat cells in the concentrations required to promote antioxidant activity in vitro, and it can be applied in topical products.

Keywords: caffeic acid, antioxidant, cytotoxicity, cosmetic

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