Search results for: IC50 and dimenazin

Authors: Latife Merve Oktay, Berrin Tugrul

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Hydroxytyrosol (HT) is a phenolic phytochemical molecule derived from the hydrolysis of oleuropein, which originates during the maturation of the olives. It has recently received particular attention because of its antioxidant, anti-proliferative, pro-apoptotic and anti-inflammatory activities. In this study, we investigated the cytotoxic and apoptotic effects of hydroxytyrosol and its effects on phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase 1/2 (ERK 1/2) signaling pathways in human ovarian cancer cell lines OVCAR-3 and MDAH-2774. XTT cell proliferation kit, Cell Death Detection Elisa Plus Kit (Roche) and Human Apoptosis Array (R&D Systems) were used to determine the cytotoxic and apoptotic effects of HT in OVCAR-3 and MDAH-2774 cell lines at 24, 48, 72, and 96 h. Effect of HT on PI3K/Akt and ERK 1/2 signaling pathways were investigated by using specific inhibitors of these pathways. IC50 values of HT were found to be 102.3 µM in MDAH-2774 cells at 72 h and 51.5 µM in OVCAR-3 cells at 96 h. Apoptotic effect of HT in MDAH-2774 cells was the highest at 50 µM at 72 h, and kept decreasing at 100 and 150 µM concentrations and was not seen at 200 µM and higher concentrations. Highest apoptotic effect was seen at 100 µM concentration in OVCAR-3 cells at 96 h, however apoptotic effect was decreased over 100 µM concentrations. According to antibody microarray results, HT increased the levels of pro-apoptotic molecules Bad, Bax, active caspase-3, Htra2/Omi by 2.0-, 1.4-, 1.2-, 4.2-fold, respectively and also increased the levels of pro-apoptotic death receptors TRAIL R1/DR4, TRAIL R2/DR5, FAS/TNFRSF6 by 2.1-, 1.7-, 1.6-fold, respectively, however, it decreased the level of Survivin by 1.6-fold which is one of the inhibitor of apoptosis protein (IAP) family in MDAH-2774 cells. In OVCAR-3 cells, HT decreased the levels of anti-apoptotic proteins Bcl-2, pro-caspase 3 by 3.1-, 8.2-fold, respectively and IAP family proteins CIAP-1, CIAP-2, XIAP, Livin, Survivin by 6.5-, 6.0-, 3.2-, 2.2-, 2.7-fold, respectively and increased the level of cytochrome-c by 1.2-fold. We have shown that HT shows its cytotoxic and apoptotic effect through inhibiting ERK 1/2 signaling pathway in both OVCAR-3 and MDAH-2774 cells. Further studies are needed to investigate molecular mechanisms and modulatory effects of hydroxytyrosol.

Keywords: apoptosis, cytotoxicity, hydroxytyrosol, ovarian cancer

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Authors: Min-Chien Su, Tzu-Hsuan Hsu, Guan-Xuan Wu, Shyh-Ming Kuo

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Lung cancer is one of the clinically challenging malignant diseases worldwide. For efficient therapeutics in cancer, combination therapy has developed to acquire a better outcome. PLK-1 was one of the major factors affecting cell mitosis in cancer cells, its inhibitor Bi6727 was proven effective in treating several different cancers namely oral cancer, colon cancer and lung cancer. Despite its low toxicity toward normal cells compared to traditional chemotherapy, it is still yet to be evaluated in detail. Livistona Chinensis (LC) is a Chinese herb that used as a traditional prescription to treat lung cancer. Due to the uncertainty of the efficacy of LC, we utilized a water extraction method to extract the Livistona Chinensis and then lyophilized into powder for further study. In this study we investigated the antiproliferation activities of Bi6727 and LC extracts (LCE) on A549 non-small lung cancer cells. The IC50 of Bi6727 and LCE on A549 are 60 nM and 0.8 mg/mL, respectively. The fluorescent staining images shown nucleolus damage in cells treated with Bi6727 and mitochondrial damage after treated with LCE. A549 cells treated with Bi6727 and LCE showed increased expression of Bax, Caspase-3 and Caspase-9 proteins from Western blot assay. LCE also inhibited A549 cells growth keeping cells at G2-M phase from cell cycle assay. Apoptosis assay results showed that LCE induced late apoptosis of A549 cells. JC-1 assay showed that the mitochondria damaged at the LCE concentration of 0.4 mg/mL. In our preliminary anti-proliferation test of combined LCE and Bi-6727 on A549 cells, we found a dramatically decrease in proliferation after treated with LCE first for 24-h and then Bi-6727 for extra 24-h. This was an important finding regarding synergistic anti-proliferation effect of these drugs, However, the usage, the application sequence of LCE and Bi-6727 on A549 cells and their related mechanisms still need to be evaluated. In summary, the drugs exerted anti-proliferation effect on A549 cells independently. We hopefully combine the usage of these two drugs will bring a different and potential outcome in treating lung cancer.

Keywords: anti-proliferation, A549, Livistona Chinensis fruit extracts, PLK-1 inhibitor

Procedia PDF Downloads 141

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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Authors: Xiaoquan Huang, Congcong Li, Tingting Wei, Changcun Bai, Na Liu, Meng Tang

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Silver is often doped on nano-titanium dioxide photocatalysts (Ag-TiO₂) by photodeposition method to improve their utilization of visible-light while increasing the toxicity of TiO₂。 However, it is not known what factors influence this toxicity and how to reduce toxicity while maintaining the maximum catalytic activity. In this study, Ag-TiO₂ photocatalysts were synthesized by the photodeposition method with different silver content (AgC) and photodeposition time (PDT). Characterization and catalytic experiments demonstrated that silver was well assembled on TiO₂ with excellent visible-light catalytic activity, and the size of silver increased with PDT. In vitro, the cell viability of lung epithelial cells A549 and BEAS-2B showed that the higher content and smaller size of silver doping caused higher toxicity. In vivo, Ag-TiO₂ catalysts with lower AgC or larger silver particle size obviously caused less pulmonary pro-inflammatory and pro-fibrosis responses. However, the visible light catalytic activity decreased with the increase in silver size. Therefore, in order to optimize the Ag-TiO₂ photocatalyst with the lowest pulmonary toxicity and highest catalytic performance, response surface methodology (RSM) was further performed to optimize the two independent variables of AgC and PDT. Visible-light catalytic activity was evaluated by the degradation rate of Rhodamine B, the antibacterial property was evaluated by killing log value for Escherichia coli, and cytotoxicity was evaluated by IC50 to BEAS-2B cells. As a result, the RSM model showed that AgC and PDT exhibited an interaction effect on catalytic activity in the quadratic model. AgC was positively correlated with antibacterial activity. Cytotoxicity was proportional to AgC while inversely proportional to PDT. Finally, the optimization values were AgC 3.08 w/w% and PDT 28 min. Under this optimal condition, the relatively high silver proportion ensured the visible-light catalytic and antibacterial activity, while the longer PDT effectively reduced the cytotoxicity. This study is of significance for the safe and efficient application of silver-doped TiO₂ photocatalysts.

Keywords: Ag-doped TiO₂, cytotoxicity, inflammtion, fibrosis, response surface methodology

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Authors: Zohreh Bayat, Dariush Minai-Tehrani

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Pseudomonas aeruginosa is a Gram-negative, rode shape and aerobic bacterium that has shown to be resistance to many antibiotics. This resistance makes the bacterium very harmful in some diseases. It can also generate diseases in any part of the gastrointestinal tract from oropharynx to rectum. P. aeruginosa has become an important cause of infection, especially in patients with compromised host defense mechanisms. One of the most important reasons that make P. aeruginosa an emerging opportunistic pathogen in patients is its ability to use various compounds as carbon sources. Lipase is an enzyme that catalyzes the hydrolysis of lipids. Most lipases act at a specific position on the glycerol backbone of lipid substrate. Some lipases are expressed and secreted by pathogenic organisms during the infection. Muscarinic antagonist used as an antispasmodic and in urinary incontinence. The drug has little effect on glandular secretion or the cardiovascular system. It does have some local anesthetic properties and is used in gastrointestinal, biliary, and urinary tract spasms. Aim: In this study the inhibitory effect of a muscarinic antagonist on lipase of P. aeruginosa was investigated. Methods: P. aeruginosa was cultured in minimal salt medium with 1% olive oil as carbon source. The cells were harvested and the supernatant, which contained lipase, was used for enzyme assay. Results: Our results showed that the drug can inhibit P. aeruginosa lipase by competitive manner. In the presence of different concentrations of the drug, the Vmax (2 mmol/min/mg protein) of enzyme did not change, while the Km raised by increasing the drug concentration. The Ki (inhibition constant) and IC50 (the half maximal inhibitory concentration) value of drug was estimated to be about 30 uM and 60 uM which determined that the drug binds to enzyme with high affinity. Maximum activity of the enzyme was observed at pH 8 in the absence and presence of muscarinic antagonist, respectively. The maximum activity of lipase was observed at 600C and the enzyme became inactive at 900C. Conclusion: The muscarinic antagonist drug could inhibit lipase of P. aeruginosa and changed the kinetic parameters of the enzyme. The drug binded to enzyme with high affinity and did not chang the optimum pH of the enzyme. Temperature did not affect the binding of drug to musmuscarinic antagonist.

Keywords: Pseudomonas aeruginosa, drug, enzyme, inhibition

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Authors: Sohan Sengupta, Arnab Pramanik, Abhrajyoti Ghosh, Maitree Bhattacharyya

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Newly emerged phyto-pathogens and multi drug resistance have been threating the world for last few decades. Actinomycetes, the most endowed group of microorganisms isolated from unexplored regions of the world may be the ultimate solution to these problems. Thus the aim of this study was to isolate several bioactive actinomycetes strains capable of producing antimicrobial secondary metabolite from Sundarbans, the only mangrove tiger land of the world. Fifty four actinomycetes were isolated and analyzed for antimicrobial activity against fifteen test organisms including three phytopathogens. Nine morphologically distinct and biologically active isolates were subjected to polyphasic identification study. 16s rDNA sequencing indicated eight isolates to reveal maximum similarity to the genus streptomyces, whereas one isolate presented only 93.57% similarity with Streptomyces albogriseolus NRRL B-1305T. Seventy-one carbon sources and twenty-three chemical sources utilization assay revealed their metabolic relatedness. Among these nine isolates three specific strains were found to have notably higher degree of antimicrobial potential effective in a broader range including phyto-pathogenic fungus. PCR base whole genome screen for PKS and NRPS genes, confirmed the occurrence of bio-synthetic gene cluster in some of the isolates for novel antibiotic production. Finally the strain SMS_SU21, which showed antimicrobial activity with MIC value of 0.05 mg ml-1and antioxidant activity with IC50 value of 0.242±0.33 mg ml-1 was detected to be the most potential one. True prospective of this strain was evaluated utilizing GC-MS and the bioactive compound responsible for antimicrobial activity was purified and characterized. Rare bioactive actinomycetes were isolated from unexplored heritage site. Diversity of the biosynthetic gene cluster for antimicrobial compound production has also been evaluated. Antimicrobial compound SU21-C has been identified and purified which is active against a broad range of pathogens.

Keywords: actinomycetes, sundarbans, antimicrobial, pks nrps, phyto-pathogens, GC-MS

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Authors: Amel Bouaziz, Seddik Khennouf, Mussa Abu Zarga, Shtayway Abdalla, Saliha Djidel, Assia Bentahar, Saliha Dahamna, Smain Amira

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The present study aimed to evaluate the hypotensive and the in vitro antioxidant activities of Crataegus azarolus L. (Rosaceae), a plant widely used as natural remedy for hypertension in folk medicine. The antioxidant potential of methanolic extract (ME)and its three fractions of Chloroform (CHE), ethyl acetate (EAE)and water (AqE) have been investigated using several assays, including the DPPH scavenging, ABTS scavenging, hydroxyl radical scavenging. Inhibition of lipid peroxidation was performed by the β-carotene bleaching assay, ferric thiocyanate method and thiobarburic acid method. Total phenolic and total flavonoid contents of the extracts were estimated using Folin-Chiocalteu reagent and AlCl3, respectively. EAE extract showed the highest polyphenolic and flavonoids contents (396,04±1.20 mg GAE/g of dry extract and 32,73 ± 0.03mg QE/g of dry extract) respectively. Similarly, this extract possessed the highest scavenging activity for DPPH radical (IC 50 = 0,006±0,0001mg /ml), ABTS radical (IC50=0.0035±0,0007 mg/ml) and hydroxyl radical(IC 50=0,283± 0.01 mg/ml). In addition, the EAE exhibited the highest antioxidant activity in the inhibition of linoleic acid/ß-carotene coupled oxidation (89,21%), lipid peroxidation in the ferric thiocyanate(FTC) method (90.13%), and thio-barbituric acid (TBA) method (74.23%). Intravenous administration of Me and EAE decreased mean arterial blood pressure, systolic and diastolic blood pressure in anesthetized rats dose-dependently, at the dose range of 0.4 to 12 mg/kg. The mean arterial blood pressure dropped by 27.58 and 39.37% for ME and EAE, respectively. In conclusion, The present study supported the significant potential to use C. azarolus by-products as a source of natural antioxidants and provides scientific justification for its traditional uses as cardio-protective and anti-hypertensive remedy.

Keywords: Crataegus azarolus, polyphenols, flavonoids, hypertension, antioxidant activity, free radicals, peroxidation

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Authors: Mei-Ling Chan, Jin-Ming Wu, Konstantin V. Kudryavtsev, Jih-Hwa Guh

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Prostate cancer is one of the most common malignant disease in men. KUD983 is an enantiomerically pure β-dipeptide derivative, which may have anti-cancer effects. In the present study, KUD983 exhibits powerful activity against hormone-refractory prostate cancer (HRPC) PC-3 and DU145 cells. The IC50 values of KUD983 in PC-3 and DU145 cells are 0.56±0.07M and 0.50±0.04 M respectively. KUD983 induced G1 arrest of the cell cycle and subsequent apoptosis associated with the down-regulation of several related proteins including cyclin D1, cyclin E and Cdk4, and the de-phosphorylation of RB. The protein expressions of nuclear and total c-Myc protein, which was able to regulate the expression of both cyclin D1 and cyclin E, were significantly suppressed by KUD983. Phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) is an important signaling pathway that influences the energy metabolism, cell cycle, proliferation, survival and apoptosis of cells, and is associated with numerous other signaling pathways. The Western Blot data revealed that KUD983 inhibited PI3K/Akt and mTOR/p70S6K/4E-BP1 pathways. The transient transfection of constitutively active myristylated Akt (myr-Akt) cDNA significantly reversed KUD983-induced caspase activation but did not abolish the suppression of mTOR/p70S6K/4E-BP1 signaling cascade indicating the presence of both Akt-dependent and -independent pathways. Moreover, KUD983-induced effect was collaborated with the down-regulation of anti-apoptotic Bcl-2 members (e.g., Bcl-2, and Mcl-1) and IAP family members (e.g., survivin). Furthermore, KUD983 induced autophagic cell death using confocal microscopic examination, investigating the level of conversion of LC3-I to LC3-II and flow cytometric detection of AVO-positive cells. Taken together, the data suggest that KUD983 is an anticancer β-dipeptide against HRPCs through the inhibition of cell proliferation and induction of apoptotic and autophagic cell death. The suppression of signaling pathways mediated by c-Myc, PI3K/Akt and mTOR/p70S6K/4E-BP1 and the collaboration with down-regulation of Mcl-1 and survivin may indicate the mechanism of KUD983 against HRPC.

Keywords: β-dipeptide, hormone-refractory prostate cancer, mTOR, PI3K/Akt

Procedia PDF Downloads 282

Authors: Rossy G. Olan Villegas, Gerardo Acosta Garcia, Aurea Bernardino Nicanor, Leopoldo Gonzalez Cruz, Humberto Ramirez Medina

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Capsicum annuum is a crop of agricultural and economic importance in Mexico and other countries. The fruit (pepper) contains bioactive components such as carotenoids, phenolic compounds and capsaicinoids that improve health. However, pepper cultivation is affected by biotic and abiotic factors that decrease yield. Some phytohormones like gibberellins and auxins induce the formation and development of fruit in several plants. In this study, we evaluated the effect of the exogenous application of phytohormones like gibberellic acid and indolbutyric acid on fruit development of jalapeno pepper plants, the protein profile of plant tissues, the accumulation of bioactive compounds and antioxidant activity in the pericarp and seeds. For that, plants were sprinkled with these phytohormones. The fruit collection for the control, indolbutyric acid and gibberellic acid treatments was 7 peppers per plant; however, for the treatment that combines indolbutyric acid and gibberellic acid, a fruit with the shortest length (1.52 ± 1.00 cm) and weight (0.41 ± 1.0 g) was collected compared to fruits of plants grown under other treatments. The length (4,179 ± 0,130 cm) and weight of the fruit (8,949 ± 0.583 g) increased in plants treated with indolbutyric acid, but these characteristics decreased with the application of GA3 (length of 3,349 ± 0.127 cm and a weight 4,429 ± 0.144 g). The content of carotenes and phenolic compounds increased in plants treated with GA3 (1,733 ± 0.092 and 1,449 ± 0.009 mg / g, respectively) or indolbutyric acid (1,164 ± 0.042 and 0.970 ± 0.003 mg / g). However, this effect was not observed in plants treated with both phytohormones (0.238 ± 0.021 and 0.218 ± 0.004 mg / g). Capsaicin content was higher in all treatments; but it was more noticeable in plants treated with both phytohormones, the value being 0.913 ± 0.001 mg / g (three times greater in amount). The antioxidant activity was measured by 3 different assays, 2,2-diphenyl-1-picrylhydrazyl (DPPH), antioxidant power of ferric reduction (FRAP) and 2,2'-Azinobis-3-ethyl-benzothiazoline-6-sulfonic acid ( ABTS) to find the minimum inhibitory concentration of the reducing radical (IC50 and EC50). Significant differences were observed from the application of the phytohormone, being the fruits treated with gibberellins, which had a greater accumulation of bioactive compounds. Our results suggest that the application of phytohormones modifies the development of fruit and its content of bioactive compounds.

Keywords: auxins, capsaicinoids, carotenoids, gibberellins

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Authors: Raviv Harris, Maggie Levy

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Natural product-based pesticides may serve as an alternative to the traditional synthetic pesticides, which have a potentially damaging effect, both to human health and for the environment. Along with plants, microorganisms are a prospective source of such biological pesticides. A unique and active strain of P. aphidis (designated isolate L12, Israel 2004), an epiphytic and non-pathogenic basidiomycete yeast, was isolated in our lab from strawberry leaves. P. aphidis L12 secretions were found to inhibit broad range of plant pathogens. This work demonstrates that metabolites isolated from the biocontrol agent P. aphidis (isolate L12) can inhibit varied fungal and bacterial phytopathogens. Biologically active metabolites were extracted from P. aphidis biomass, using the organic solvent ethyl acetate. The antimicrobial activity of the extract was demonstrated, both in vitro and in planta. Using disk diffusion assays, the following inhibition zones were obtained: 43cm² for Pseudomonas syringae pv. tomato, 28.5cm² for Xanthomonas campestris pv. vesicatoria, 59cm² for Clavibacter michiganensis subsp. michiganensis, 34cm² for Erwinia amylovora and 34cm² for Agrobacterium tumefaciens. Additionally, strong inhibitory activity of the extract against fungi mycelial growth was established, with IC₅₀ values of 606µg ml⁻¹ for Botrytis cinerea, 221µg ml⁻¹ for Pythium spp., 519µg ml⁻¹ for Rhizoctonia solani, 455µg ml⁻¹ for Sclerotinia sclerotiorum, 2270µg ml⁻¹ for Fusarium oxysporum f. sp. lycopersici, and 2038µg ml⁻¹ for Alternaria alternata. The results of the in planta experiments demonstrated a dose-dependent reduction in disease infection. Significant inhibition of B. cinerea lesions on tomato plants was obtained when a spore suspension of this pathogen was treated with extract concentrations higher than 4.2mg ml⁻¹. Concentration of 7mg ml⁻¹ caused a reduction of over 95% in the lesion size of B. cinerea on tomato plants. The strong antimicrobial activity demonstrated both in vitro and in planta against varied phytopathogens, may indicate that the extracted antimicrobial metabolites have potential to serve as natural pesticides in the field.

Keywords: antimicrobial, B. cinerea, metabolites, natural pesticides, P. aphidis

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Authors: Karol Rodriguez-Peña, Martha L. Macias-Rubalcava, Leticia Rocha-Zavaleta, Sergio Sanchez

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A bioassay-guided study for anti-cancer compounds from endophytes of the Mexican medicinal plant Amphipteryygium adstringens resulted in the isolation of a streptomycete capable of producing a group of compounds with high cytotoxic activity. Microorganisms from surface sterilized samples of various sections of the plant were isolated and all the actinomycetes found were evaluated for their potential to produce compounds with cytotoxic activity against cancer cell lines MCF7 (breast cancer) and HeLa (cervical cancer) as well as the non-tumoural cell line HaCaT (keratinocyte). The most active microorganism was picked for further evaluation. The identification of the microorganism was carried out by 16S rDNA gene sequencing, finding the closest proximity to Streptomyces scabrisporus, but with the additional characteristic that the strain isolated in this study was capable of producing colorful compounds never described for this species. Crude extracts of dichloromethane and ethyl acetate showed IC50 values of 0.29 and 0.96 μg/mL for MCF7, 0.51 and 1.98 μg/mL for HeLa and 0.96 and 2.7 μg/mL for HaCaT. Scaling the fermentation to 10 L in a bioreactor generated 1 g of total crude extract, which was fractionated by silica gel open column to yield 14 fractions. Nine of the fractions showed cytotoxic activity. Fraction 4 was chosen for subsequent purification because of its high activity against cancerous cell lines, lower activity against keratinocytes. HPLC-UV-MS/ESI was used for the evaluation of this fraction, finding at least 10 different compounds with high values of m/z (≈588). Purification of the compounds was carried out by preparative thin-layer chromatography. The prevalent compound was Steffimycin B, a molecule known for its antibiotic and cytotoxic activities and also for its low solubility in aqueous solutions. Along with steffimycin B, another five compounds belonging to the steffimycin family were isolated and at this moment their structures are being elucidated, some of which display better solubility in water: an attractive property for the pharmaceutical industry. As a conclusion to this study, the isolation of endophytes resulted in the discovery of a strain capable of producing compounds with high cytotoxic activity that need to be studied for their possible utilization.

Keywords: amphipterygium adstringens, cytotoxicity, streptomyces scabrisporus, steffimycin

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Authors: Richa Dhingra, Monika, Manav Malhotra, Tilak Raj Bhardwaj, Neelima Dhingra

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5-Alpha-reductases (5AR), a membrane bound, NADPH dependent enzyme and convert male hormone testosterone (T) into more potent androgen dihydrotestosterone (DHT). DHT is the required for the development and function of male sex organs, but its overproduction has been found to be associated with physiological conditions like Benign Prostatic Hyperplasia (BPH). Thus the inhibition of 5ARs could be a key target for the treatment of BPH. In present study, 2D and 3D Quantitative Structure Activity Relationship (QSAR) pharmacophore models have been generated for 5AR based on known inhibitory concentration (IC₅₀) values with extensive validations. The four featured 2D pharmacophore based PLS model correlated the topological interactions (–OH group connected with one single bond) (SsOHE-index); semi-empirical (Quadrupole2) and physicochemical descriptors (Mol. wt, Bromines Count, Chlorines Count) with 5AR inhibitory activity, and has the highest correlation coefficient (r² = 0.98, q² =0.84; F = 57.87, pred r² = 0.88). Internal and external validation was carried out using test and proposed set of compounds. The contribution plot of electrostatic field effects and steric interactions generated by 3D-QSAR showed interesting results in terms of internal and external predictability. The well validated 2D Partial Least Squares (PLS) and 3D k-nearest neighbour (kNN) models were used to search novel 5AR inhibitors with different chemical scaffold. To gain more insights into the molecular mechanism of action of these steroidal derivatives, molecular docking and in silico absorption, distribution, metabolism, and excretion (ADME) studies were also performed. Studies have revealed the hydrophobic and hydrogen bonding of the ligand with residues Alanine (ALA) 63A, Threonine (THR) 60A, and Arginine (ARG) 456A of 4AT0 protein at the hinge region. The results of QSAR, molecular docking, in silico ADME studies provide guideline and mechanistic scope for the identification of more potent 5-Alpha-reductase inhibitors (5ARI).

Keywords: 5α-reductase inhibitor, benign prostatic hyperplasia, ligands, molecular docking, QSAR

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Authors: Rachmat Mauludin, Dita Sasri Primaviri, Irda Fidrianny

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Propolis contains several antioxidant compounds which can be used in topical application to protect skin against free radical, prevent skin cancer and skin aging. Previous study showed that 70% ethanolic extract of propolis (EEP) provided the greatest antioxidant activity. Since EEP has very small solubility in water, the extract was prepared in nanoemulsion (NE). Nanoemulsion is chosen as cosmetic dosage forms according to its properties namely to decrease the risk of skin’s irritation, increase penetration, prolong its time to remain in our skin, and improve stability. Propolis was extracted using reflux methods and concentrated using rotavapor. EEP was characterized with several tests such as phytochemical screening, density, and antioxidant activity using DPPH method. Optimation of total surfactant, co-surfactant, oil, and amount of EEP that can be included in NE were required to get the best NE formulation. The evaluations included to organoleptic observation, globul size, polydispersity index, morphology using TEM, viscosity, pH, centrifuge, stability, Freeze and Thaw test, radical scavenging activity using DPPH method, and primary irritation test. The yield extracts was 11.12% from raw propolis contained of steroid/triterpenoid, flavonoid, and saponin based on phytochemical screening. EEP had the value of DPPH scavenging activity 61.14% and IC50 0.41629 ppm. The best NE formulation consisted of 26.25% Kolliphor RH40; 8.75% glycerine; 5% rice bran oil; and 3% EEP. NE was transparant, had globul size of 21.9 nm; polydispersity index of 0.338; and pH of 5.67. Based on TEM morphology, NE was almost spherical and has particle size below 50 nm. NE propolis revealed to be physically stable after stability test within 63 days at 25oC, centrifuged for 30 mins at 13.000 rpm, and passed 6 cycles of Freeze and Thaw test without separated. NE propolis reduced 58% of free radical DPPH similar to antioxidant activity of the original extracts. Antioxidant activity of NE propolis is relatively stable after stored for 6 weeks. NE Propolis was proven to be safe by primary irritation test with the value of primary irritation index (OECD) was 0. The best formulation for NE propolis contained of 26.25% Kolliphor RH40; 8.75% glycerine; 5% rice bran oil; and 3% EEP with globul size of 21.9 nm and polydispersity index of 0.338. NE propolis was stable and had antioxidant activity similar to EEP.

Keywords: propolis, antioxidant, nanoemulsion, irritation test

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Authors: Rachmat Mauludin, Irda Fidrianny, Dita Sasri Primaviri, Okti Alifiana

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Natural products such as propolis, green tea and corncob are containing several compounds called antioxidant. Antioxidant can be used in topical application to protect skin against free radical, prevent skin cancer and skin aging. Previous study showed that the extract of propolis that has the highest antioxidant activity was ethanolic extract of propolis (EEP). It is important to make a dosage form that could keep the stability and could protect the effectiveness of antioxidant activity of the extracts. In this research, nanoemulsion (NE) was chosen to formulate those natural products. NE is a dispersion system between oil phase and water phase that formed by mechanical force with a lot amount of surfactants and has globule size below 100 nm. In pharmaceutical industries, NE was preferable for its stability, biodegradability, biocompatibility, its ease to be absorbed and eliminated, and for its use as carrier for lipophilic drugs. First, all of the natural products were extracted using reflux methods. Green tea and corncob were extracted using 96% ethanol while propolis using 70% ethanol. Then, the extracts were concentrated using rotavapor to obtain viscous extracts. The yield of EEP was 11.12%; green tea extract (GTE) was 23.37%; and corncob extract (CCE) was 17.23%. EEP contained steroid/triterpenoid, flavonoid and saponin. GTE contained flavonoid, tannin, and quinone while CCE contained flavonoid, phenol and tannin. The antioxidant activities of the extracts were then measured using DPPH scavenging capacity methods. The values of DPPH scavenging capacity were 61.14% for EEP; 97.16% for GTE; and 78.28% for CCE. The value of IC50 for EEP was 0.41629 ppm. After the extracts were evaluated, NE was prepared. Several surfactants and co-surfactants were used in many combinations and ratios in order to form a NE. Tween 80 and Kolliphor RH40 were used as surfactants while glycerin and propylene glycol were used as co-surfactants. The best NE consists of 26.25% of Kolliphor RH40; 8.75% of glycerin; 5% of rice bran oil; 3% of extracts; and 57% of water. EEP NE had globule size around 23.72 nm; polydispersity index below 0.5; and did not cause any irritation on rabbits. EEP NE was proven to be stable after passing stability test within 63 days at room temperature and 6 cycles of Freeze and Thaw test without separated. Based on TEM (Transmission Electron Microscopy) test, EEP NE had spherical structure with most of its size below 50 nm. The antioxidant activity of EEP NE was monitored for 6 weeks and showed no significant difference. The value of DPPH scavenging capacity for EEP NE was around 58%; for GTE NE was 96.75%; and for CCE NE was 55.69%.

Keywords: propolis, green tea, corncob, antioxidant, nanoemulsion

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Authors: Emma R. Arakelova, Stepan G. Grigoryan, Flora G. Arsenyan, Nelli S. Babayan, Ruzanna M. Grigoryan, Natalia K. Sarkisyan

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Novel nanosize zinc oxide composites of doxorubicin obtained by deposition of 180 nm thick zinc oxide film on the drug surface using DC-magnetron sputtering of a zinc target in the form of gels (PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO) were studied for drug delivery applications. The cancer specificity was revealed both in in vitro and in vivo models. The cytotoxicity of the test compounds was analyzed against human cancer (HeLa) and normal (MRC5) cell lines using MTT colorimetric cell viability assay. IC50 values were determined and compared to reveal the cancer specificity of the test samples. The mechanistic study of the most active compound was investigated using Flow cytometry analyzing of the DNA content after PI (propidium iodide) staining. Data were analyzed with Tree Star FlowJo software using cell cycle analysis Dean-Jett-Fox module. The in vivo anticancer activity estimation experiments were carried out on mice with inoculated ascitic Ehrlich’s carcinoma at intraperitoneal introduction of doxorubicin and its zinc oxide compositions. It was shown that the nanosize zinc oxide film deposition on the drug surface leads to the selective anticancer activity of composites at the cellular level with the range of selectivity index (SI) from 4 (Starch+NaCMC+Dox+ZnO) to 200 (PEO(gel)+Dox+ZnO) which is higher than that of free Dox (SI = 56). The significant increase in vivo antitumor activity (by a factor of 2-2.5) and decrease of general toxicity of zinc oxide compositions of doxorubicin in the form of the above mentioned gels compared to free doxorubicin were shown on the model of inoculated Ehrlich's ascitic carcinoma. Mechanistic studies of anticancer activity revealed the cytostatic effect based on the high level of DNA biosynthesis inhibition at considerable low concentrations of zinc oxide compositions of doxorubicin. The results of studies in vitro and in vivo behavior of PEO+Dox+ZnO and Starch+NaCMC+Dox+ZnO composites confirm the high potential of the nanosize zinc oxide composites as a vector delivery system for future application in cancer chemotherapy.

Keywords: anticancer activity, cancer specificity, doxorubicin, zinc oxide

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Authors: Adebisi Adunola Demehin, Wanlaya Thamnarak, Jaruwan Chatwichien, Chatchakorn Eurtivong, Kiattawee Choowongkomon, Somsak Ruchirawat, Nopporn Thasana

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The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein involved in cellular signalling processes and, its aberrant activity is crucial in the development of many cancers such as lung cancer. Selaginellaceae are fern allies that have long been used in Chinese traditional medicine to treat various cancer types, especially lung cancer. Biflavonoids, the major secondary metabolites in Selaginellaceae, have numerous pharmacological activities, including anti-cancer and anti-inflammatory. For instance, amentoflavone induces a cytotoxic effect in the human NSCLC cell line via the inhibition of PARP-1. However, to the best of our knowledge, there are no studies on biflavonoids as EGFR inhibitors. Thus, this study aims to investigate the EGFR inhibitory activities of biflavonoids isolated from Selaginella siamensis and Selaginella bryopteris. Amentoflavone, tetrahydroamentoflavone, sciadopitysin, robustaflavone, robustaflavone-4-methylether, delicaflavone, and chrysocauloflavone were isolated from the ethyl-acetate extract of the whole plants. The structures were determined using NMR spectroscopy and mass spectrometry. In vitro study was conducted to evaluate their cytotoxicity against A549, HEPG2, and T47D human cancer cell lines using the MTT assay. In addition, a target-based assay was performed to investigate their EGFR inhibitory activity using the kinase inhibition assay. Finally, a molecular docking study was conducted to predict the binding modes of the compounds. Robustaflavone-4-methylether and delicaflavone showed the best cytotoxic activity on all the cell lines with IC50 (µM) values of 18.9 ± 2.1 and 22.7 ± 3.3 on A549, respectively. Of these biflavonoids, delicaflavone showed the most potent EGFR inhibitory activity with an 84% relative inhibition at 0.02 nM using erlotinib as a positive control. Robustaflavone-4-methylether showed a 78% inhibition at 0.15 nM. The docking scores obtained from the molecular docking study correlated with the kinase inhibition assay. Robustaflavone-4-methylether and delicaflavone had a docking score of 72.0 and 86.5, respectively. The inhibitory activity of delicaflavone seemed to be linked with the C2”=C3” and 3-O-4”’ linkage pattern. Thus, this study suggests that the structural features of these compounds could serve as a basis for developing new EGFR-TK inhibitors.

Keywords: anticancer, biflavonoids, EGFR, molecular docking, Selaginellaceae

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Authors: Bagnoud-Velásquez Mariluz, Damergi Eya, Grandjean Dominique, Frédéric Vogel, Ludwig Christian

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The sustainability of algae to biofuel processes is seriously affected by the energy intensive production of fertilizers. Large amounts of nitrogen and phosphorus are required for a large-scale production resulting in many cases in a negative impact of the limited mineral resources. In order to meet the algal bioenergy opportunity it appears crucial the promotion of processes applying a nutrient recovery and/or making use of renewable sources including waste. Hydrothermal (HT) conversion is a promising and suitable technology for microalgae to generate biofuels. Besides the fact that water is used as a “green” reactant and solvent and that no biomass drying is required, the technology offers a great potential for nutrient recycling. This study evaluated the possibility to treat the water HT effluent by the growth of microalgae while producing renewable algal biomass. As already demonstrated in previous works by the authors, the HT aqueous product besides having N, P and other important nutrients, presents a small fraction of organic compounds rarely studied. Therefore, extracted heteroaromatic compounds in the HT effluent were the target of the present research; they were profiled using GC-MS and LC-MS-MS. The results indicate the presence of cyclic amides, piperazinediones, amines and their derivatives. The most prominent nitrogenous organic compounds (NOC’s) in the extracts were carefully examined by their effect on microalgae, namely 2-pyrrolidinone and β-phenylethylamine (β-PEA). These two substances were prepared at three different concentrations (10, 50 and 150 ppm). This toxicity bioassay used three different microalgae strains: Phaeodactylum tricornutum, Chlorella sorokiniana and Scenedesmus vacuolatus. The confirmed IC50 was for all cases ca. 75ppm. Experimental conditions were set up for the growth of microalgae in the aqueous phase by adjusting the nitrogen concentration (the key nutrient for algae) to fit that one established for a known commercial medium. The values of specific NOC’s were lowered at concentrations of 8.5 mg/L 2-pyrrolidinone; 1mg/L δ-valerolactam and 0.5 mg/L β-PEA. The growth with the diluted HT solution was kept constant with no inhibition evidence. An additional ongoing test is addressing the possibility to apply an integrated water cleanup step making use of the existent hydrothermal catalytic facility.

Keywords: hydrothermal process, microalgae, nitrogenous organic compounds, nutrient recovery, renewable biomass

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Authors: Yohannes Kelifa, Gomathi Periasamy, Aman Karim

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Introduction: Diabetes mellitus has become a major public health and economical problem across the globe. Modern antidiabetic drugs have a number of limitations, and scientific investigation of traditional herbal remedies used for diabetes may provide novel leads for the development of new antidiabetic drugs that can be used as alternative or complementary to available antidiabetic allopathic medications. Though Myrica salicifolia Hochst. ex A. Rich. is used for the management of diabetes in Ethiopian traditional medicine, there was no previous scientific evidence about its antidiabetic effect to the authors’ knowledge. This study was undertaken to evaluate the antidiabetic activity the root extracts of Myrica salicifolia in streptozotocin (STZ)-induced diabetic mice. Methods: Experimental diabetes was induced by intraperitoneal administration of STZ (150 mg/kg) in male mice. Diabetic mice were treated with oral doses of M. salicifolia root extracts at 200, 400 and 600 mg/kg, and its fractions (chloroform, ethyl acetate, n-butanol and aqueous) at a dose of 400 mg/kg daily for 15 days. Fasting blood glucose level (BGL) was measured at 0, 5th,10th, and 15th day. The free radical scavenging activity of the crude extract was determined using in vitro by DPPH assay. The statistical significance was assessed by one-way ANOVA, followed by Tukey’s multiple comparison tests. Results were considered significant when p < 0.05. Results: Daily administration of the M. salicifolia 80% methanol root extracts (at three different doses (200, 400 and 600 mg/kg) significantly (p < 0.05, p < 0.01 and p < 0.001) reduced fasting BGL compared with diabetic control. The aqueous and butanol fractions at a dose of 400 mg/kg resulted in maximum reduction of fasting BGL by 42.39%, and 52.13%, respectively at the 15th day in STZ-induced diabetic mice. Free radical scavenging activity of the 80% methanol extract of M. salicifolia was comparable to ascorbic acid. The IC50 values of the crude extract and ascorbic acid (a reference compound) were found to be 4.54 μg/ml and 4.39 μg/ml, respectively. Conclusion: These findings demonstrated that the methanolic extracts of M. salicifolia root and its fractions (n-butanol and aqueous) exhibit a significant antihyperglycemic activity in STZ-induced diabetic mice. Furthermore, the result of the present study indicates that M. salicifolia root extract is a potential source of natural antioxidants.

Keywords: antidiabetic, diabetes mellitus, DPPH, mice, Myrica salicifolia, streptozotocin

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Authors: Sharat Chandra, Zilong Wang, Ru-Rong Ji, Andrey Bortsov

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Chronic pain (CP) therapeutic approaches have limited efficacy. As a result, doctors are prescribing opioids for chronic pain, leading to opioid overuse, abuse, and addiction epidemic. Therefore, the development of effective and safe CP drugs remains an unmet medical need. Voltage-gated sodium (Nav) channels act as cardiovascular and neurological disorder’s molecular targets. Nav channels selective inhibitors are hard to design because there are nine closely-related isoforms (Nav1.1-1.9) that share the protein sequence segments. We are targeting the Nav1.7 found in the peripheral nervous system and engaged in the perception of pain. The objective of this project was to screen a 1.5 million compound library for identification of inhibitors for Nav1.7 with analgesic effect. In this study, we designed a protocol for identification of isoform-selective inhibitors of Nav1.7, by utilizing the prior information on isoform-selective antagonists. First, a similarity search was performed; then the identified hits were docked into a binding site on the fourth voltage-sensor domain (VSD4) of Nav1.7. We used the FTrees tool for similarity searching and library generation; the generated library was docked in the VSD4 domain binding site using FlexX and compounds were shortlisted using a FlexX score and SeeSAR hyde scoring. Finally, the top 25 compounds were tested with molecular dynamics simulation (MDS). We reduced our list to 9 compounds based on the MDS root mean square deviation plot and obtained them from a vendor for in vitro and in vivo validation. Whole-cell patch-clamp recordings in HEK-293 cells and dorsal root ganglion neurons were conducted. We used patch pipettes to record transient Na⁺ currents. One of the compounds reduced the peak sodium currents in Nav1.7-HEK-293 stable cell line in a dose-dependent manner, with IC50 values at 0.74 µM. In summary, our computer-aided analgesic discovery approach allowed us to develop pre-clinical analgesic candidate with significant reduction of time and cost.

Keywords: chronic pain, voltage-gated sodium channel, isoform-selective antagonist, similarity search, virtual screening, analgesics development

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Authors: Amarachi Delight Onyemachi, Gregory Ikechukwu Onwuka

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The growing consumer trend for natural ingredients, functional foods with health benefits and the perceived risk of carcinogenesis associated with synthetic antioxidants have forced food manufacturers to look for alternatives for producing healthy and safe food. Herbs and spices are cheap, natural and harmless sources of antioxidants which can delay and prevent lipid oxidation of food products and also confer its unique organoleptic properties and health benefits to food products. The Nigerian climate has been proven to be conducive for the production of spices and herbs and is blessed bountifully with a wide range of them. Five selected Nigerian herbs and spices Piper guieense, Xylopia aethopica, Gongronema latifolium and Ocimum gratissimum were evaluated for their ability to act as radical scavengers. The spices were extracted with 80% ethanol and evaluated using total phenolic capacity (TPC), DPPH (1,1-diph diphenyl-2-picrylhydrazyl radical) ABTS (2,2’azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)), total antioxidant capacity (TAC), reducing power (RP) assays. The TPC ranged from 5.33 µg GAE/mg (in Gongronema latifolium) to 15.55 µg GAE/mg (in Ocimum gratissimum). The DPPH and ABTS scavenging activity of the extracts ranged from 0.23-0.36 IC50 mg/ml and 2.32-7.25 Trolox equivalent % respectively. The TAC and RP of the extract ranged from 6.73-10.64 µg AAE/mg and 3.52-10.19 µg AAE/mg. The result of percentage yield of the extract ranged from as low as 9.94% in Gongronema latifolium and to as high as 23.85% in Xylopia aethopica. A very strong positive relationship existed between the total antioxidant capacity and total phenolic content of the tested herbs and spices (R2=0.96). All of the extracts exhibited different extent of strong antioxidant activity, high antioxidant activity was found in Ocimum gratissimum and Gongronema latifolium with the least. However, Gongronema latifolium possessed the highest total antioxidant capacity. These data confirm the appreciable antioxidant potentials and high phenolic content of Nigerian herbs and spices, thereby providing justification for their use in dishes and functional foods, prevention of cellular damage caused by free radicals and use as natural antioxidants in the food industry for prevention of lipid oxidation in food products. However, to utilize these natural antioxidants in food products, further analysis and studies of their behaviour in food systems at varying temperature, pH conditions and ionic concentrations should be carried out to displace the use of synthetic antioxidants like BHT and BHA.

Keywords: Antioxidant, free radicals, herbs, phenolic, spices

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Authors: Anik Barua, Rabiul Hossain, Labonno Barua, Rashadul Hossain, Nurul Absar

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Background: Globally, the burden of cancer is increasing consistently. Modern cancer therapies include lots of toxicity in the non-targeted organs reducing the life expectancy of the patients. Hence, scientists are trying to seek noble compounds from natural sources to treat cancer. Objectives: The objectives of the present study are to evaluate the phytochemicals, in vitro antioxidants, and in vivo and in silico anticancer study of various solvent fractions of Tinospora cordifolia (Willd.). Methodology: In this experiment, standard quantitative and qualitative assay methods were used to analyze the phytochemicals. The antioxidant activity was measured using the DPPH and ABTS scavenging methods. The in vivo antitumor activity is evaluated against Ehrlich ascites carcinoma (EAC) cell bearing in Swiss albino mice. In-silico ADME/T and molecular docking study were performed to assess the potential of stated phytochemicals against Transcription Factor STAT3b/DNA Complex of adenocarcinoma. Findings: Phytochemical screening confirmed the presence of flavonoids, alkaloids, glycosides, tannins, and carbohydrates. A significant amount of phenolic (20.19±0.3 mg/g GAE) and flavonoids (9.46±0.18 mg/g GAE) were found in methanolic extract in quantitative screening. Tinospora cordifolia methanolic extract showed promising DPPH and ABTS scavenging activity with the IC50 value of 1222.99 µg/mL and 1534.34 µg/mL, respectively, which was concentration dependent. In vivo anticancer activity in EAC cell-bearing mice showed significant (P < 0.05) percent inhibition of cell growth (60.12±1.22) was found at the highest dose compared with standard drug 5-Fluorouracil (81.18±1.28). Forty-two phytochemicals exhibit notable pharmacokinetics properties and passed drug-likeness screening tests in silico. In molecular docking study, (25S)-3Beta-acetoxy-5-alpha-22-beta-spirost-9(11)-en-12-beta-ol showed docking score (-8.5 kJ/mol) with significant non-bonding interactions with target enzyme. Conclusions: The results were found to be significant and confirmed that the methanolic extract of Tinospora cordifolia has remarkable antitumor activity with antioxidant potential. The Tinospora cordifolia methanolic extract may be considered a potent anticancer agent for advanced research.

Keywords: anticancer, antioxidant, Tinospora cordifolia, EAC cell

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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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Authors: Caroline Mendes, Mary McNamara, Orla Howe

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For chemotherapy, a drug delivery system should be able to specifically target cancer cells and deliver the therapeutic dose without affecting normal cells. Folate receptors (FR) can be considered key targets since they are commonly over-expressed in cancer cells and they are the molecular marker used in this study. Here, cyclodextrin (CD) has being studied as a vehicle for delivering the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within the CD cavity. In this study, β-CD has been modified using folic acid so as to specifically target the FR molecular marker. Thus, the system studied here for drug delivery consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 9.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 10.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 8.5 for A549 and 132.6 µM ± 12.1 and 288.1 µM ± 16.3 for BEAS-2B. These results demonstrate that MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug.

Keywords: cyclodextrins, cancer treatment, drug delivery, folate receptors, reduced folate carriers

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Authors: Ginpreet Kaur, Mohammad Yasir Usmani, Mohammed Kamil Khan

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Diabetes mellitus is a disease with a high global burden and results in significant morbidity and mortality. In India, the number of people suffering with diabetes is expected to rise from 19 to 57 million in 2025. At present, interest in herbal remedies is growing to reduce the side effects associated with conventional dosage form like oral hypoglycemic agents and insulin for the treatment of diabetes mellitus. Our aim was to investigate the antidiabetic activities of combinatorial extract of N. sativa & C. cassia in Streptozotocin induced type-I Diabetic Rats. Thus, the present study was undertaken to screen postprandial glucose excursion potential through α- glucosidase inhibitory activity (In Vitro) and effect of combinatorial extract of N. sativa & C. cassia in Streptozotocin induced type-I Diabetic Rats (In Vivo). In addition changes in body weight, plasma glucose, lipid profile and kidney profile were also determined. The IC50 values for both extract and Acarbose was calculated by extrapolation method. Combinatorial extract of N. sativa & C. cassia at different dosages (100 and 200 mg/kg orally) and Metformin (50 mg/kg orally) as the standard drug was administered for 28 days and then biochemical estimation, body weights and OGTT (Oral glucose tolerance test) were determined. Histopathological studies were also performed on kidney and pancreatic tissue. In In-Vitro the combinatorial extract shows much more inhibiting effect than the individual extracts. The results reveals that combinatorial extract of N. sativa & C. cassia has shown significant decrease in plasma glucose (p<0.0001), total cholesterol and LDL levels when compared with the STZ group The decreasing level of BUN and creatinine revealed the protection of N. sativa & C. cassia extracts against nephropathy associated with diabetes. Combination of N. sativa & C. cassia significantly improved glucose tolerance to exogenously administered glucose (2 g/kg) after 60, 90 and 120 min interval on OGTT in high dose streptozotocin induced diabetic rats compared with the untreated control group. Histopathological studies shown that treatment with N. sativa & C. cassia extract alone and in combination restored pancreatic tissue integrity and was able to regenerate the STZ damaged pancreatic β cells. Thus, the present study reveals that combination of N. sativa & C. cassia extract has significant α- glucosidase inhibitory activity and thus has great potential as a new source for diabetes treatment.

Keywords: lipid levels, OGTT, diabetes, herbs, glucosidase

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Authors: Niharika Kaushal, Minni Singh

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Citrus fruits contain an abundance of phytochemicals that can promote health. A substantial amount of agrowaste is produced from the juice processing industries, primarily peels and seeds. This leftover agrowaste is a reservoir of nutraceuticals, particularly bioflavonoids which render it antioxidant and potentially anticancerous. It is, therefore, favorable to utilize this biomass and contribute towards sustainability in a manner that value-added products may be derived from them, nutraceuticals, in this study. However, the pre-systemic metabolism of flavonoids in the gastric phase limits the effectiveness of these bioflavonoids derived from mandarin biomass. In this study, ‘kinnow’ mandarin (Citrus nobilis X Citrus deliciosa) biomass was explored for its flavonoid profile. This work entails supercritical fluid extraction and identification of bioflavonoids from mandarin biomass. Furthermore, to overcome the limitations of these flavonoids in the gastrointestinal tract, a double-layered vehicular mechanism comprising the fabrication of nanoconjugates and edible hydrogels was adopted. Total flavonoids in the mandarin peel extract were estimated by the aluminum chloride complexation method and were found to be 47.3±1.06 mg/ml rutin equivalents as total flavonoids. Mass spectral analysis revealed the abundance of polymethoxyflavones (PMFs), nobiletin and tangeretin as the major flavonoids in the extract, followed by hesperetin and naringenin. Furthermore, the antioxidant potential was analyzed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, which showed an IC50 of 0.55μg/ml. Nanoconjugates were fabricated via the solvent evaporation method, which was further impregnated into hydrogels. Additionally, the release characteristics of nanoconjugate-laden hydrogels in a simulated gastrointestinal environment were studied. The PLGA-PMFs nanoconjugates exhibited a particle size between 200-250nm having a smooth and spherical shape as revealed by FE-SEM. The impregnated alginate hydrogels offered a dense network that ensured the holding of PLGA-PMF nanoconjugates, as confirmed by Cryo-SEM images. Rheological studies revealed the shear-thinning behavior of hydrogels and their high resistance to deformation. Gastrointestinal studies showed a negligible 4.0% release of flavonoids in the gastric phase, followed by a sustained release over the next hours in the intestinal environment. Therefore, based on the enormous potential of recovering nutraceuticals from agro-processing wastes, further augmented by nanotechnological interventions for enhancing the bioefficacy of these compounds, lays the foundation for exploring the path towards the development of value-added products, thereby contributing towards the sustainable use of agrowaste.

Keywords: agrowaste, gastrointestinal, hydrogel, nutraceuticals

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Authors: Sarah Crawford, Gerry Lesley

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This research is a pre-clinical assessment of anti-cancer effects of novel non-steroidal diethyl stilbestrol-like estrogen analogs in estrogen-receptor positive/ progesterone-receptor positive human breast cancer microtumors of MCF 7 cell line. Tamoxifen analog formulation (Tam A1) was used as a single agent or in combination with therapeutic concentrations of 4-hydroxytamoxifen, currently used as a long-term treatment for the prevention of breast cancer recurrence in women with estrogen receptor positive/ progesterone receptor positive malignancies. At concentrations ranging from 30-50 microM, Tam A1 induced microtumor disaggregation and cell death. Incremental cytotoxic effects correlated with increasing concentrations of Tam A1. Live tumor microscopy showed that microtumos displayed diffuse borders and substrate-attached cells were rounded-up and poorly adherent. A complete cytotoxic effect was observed using 40-50 microM Tam A1 with time course kinetics similar to 4-hydroxytamoxifen. Combined treatment with TamA1 (30-50 microM) and 4-hydroxytamoxifen (10-15 microM) induced a highly cytotoxic, synergistic combined treatment response that was more rapid and complete than using 4-hydroxytamoxifen as a single agent therapeutic. Microtumors completely dispersed or formed necrotic foci indicating a highly cytotoxic combined treatment response. Moreover, breast cancer microtumors treated with both 4-hydroxytamoxifen and Tam A1 displayed lower levels of long-term post-treatment regrowth, a critical parameter of primary drug resistance, than observed for 4-hydroxytamoxifen when used as a single agent therapeutic. Tumor regrowth at 6 weeks post-treatment with either single agent 4-hydroxy tamoxifen, Tam A1 or a combined treatment was assessed for the development of drug resistance. Breast cancer cells treated with both 4-hydroxytamoxifen and Tam A1 displayed significantly lower levels of post-treatment regrowth, indicative of decreased drug resistance, than observed for either single treatment modality. The preclinical data suggest that combined treatment involving the use of tamoxifen analogs may be a novel clinical approach for long-term maintenance therapy in patients with estrogen-receptor positive/progesterone-receptor positive breast cancer receiving hormonal therapy to prevent disease recurrence. Detailed data on time-course, IC50 and tumor regrowth assays post- treatment as well as a proposed mechanism of action to account for observed synergistic drug effects will be presented.

Keywords: 4-hydroxytamoxifen, tamoxifen analog, drug-resistance, microtumors

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Authors: Caroline Mendes, Mary McNamara, Orla Howe

Abstract:

Drug delivery systems are proposed for use in cancer treatment to specifically target cancer cells and deliver a therapeutic dose without affecting normal cells. For that purpose, the use of folate receptors (FR) can be considered a key strategy, since they are commonly over-expressed in cancer cells. In this study, cyclodextrins (CD) have being used as vehicles to target FR and deliver the chemotherapeutic drug, methotrexate (MTX). CDs have the ability to form inclusion complexes, in which molecules of suitable dimensions are included within their cavities. Here, β-CD has been modified using folic acid so as to specifically target the FR. Thus, this drug delivery system consists of β-CD, folic acid and MTX (CDEnFA:MTX). Cellular uptake of folic acid is mediated with high affinity by folate receptors while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the cancer cell lines CaCo-2, SKOV-3, HeLa, MCF-7, A549 and the normal cell line BEAS-2B, quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. From that, four cell lines with different levels of FRs, were chosen for cytotoxicity assays of MTX and CDEnFA:MTX using the MTT assay. Real-time PCR and flow cytometry data demonstrated that all cell lines ubiquitously express moderate levels of RFC. These experiments have also shown that levels of FR protein in CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. FRs are highly expressed in all the cancer cell lines analysed when compared to the normal cell line BEAS-2B. The cell lines CaCo-2, MCF-7, A549 and BEAS-2B were used in the cell viability assays. 48 hours treatment with the free drug and the complex resulted in IC50 values of 93.9 µM ± 15.2 and 56.0 µM ± 4.0 for CaCo-2 for free MTX and CDEnFA:MTX respectively, 118.2 µM ± 16.8 and 97.8 µM ± 12.3 for MCF-7, 36.4 µM ± 6.9 and 75.0 µM ± 10.5 for A549 and 132.6 µM ± 16.1 and 288.1 µM ± 26.3 for BEAS-2B. These results demonstrate that free MTX is more toxic towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, these results demonstrate that the inclusion complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug. The use of cell imaging by confocal microscopy has allowed visualisation of FR targeting in cancer cells, as well as the identification of the interlisation pathway of the drug. Hence, the cellular uptake and internalisation process of this drug delivery system is being addressed.

Keywords: cancer treatment, cyclodextrins, drug delivery, folate receptors, reduced folate carriers

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Authors: Niharika Kaushal, Minni Singh

Abstract:

Fruit crops are widely cultivated throughout the World, with citrus being one of the most common. Mandarins, oranges, grapefruits, lemons, and limes are among the most frequently grown varieties. Citrus cultivars are industrially processed into juice, resulting in approx. 25-40% by wt. of biomass in the form of peels and seeds, generally considered as waste. In consequence, a significant amount of this nutraceutical-enriched biomass goes to waste, which, if utilized wisely, could revolutionize the functional food industry, as this biomass possesses a wide range of bioactive compounds, mainly within the class of polyphenols and terpenoids, making them an abundant source of functional bioactive. Mandarin is a potential source of bioflavonoids with putative antioxidative properties, and its potential application for developing value-added products is obvious. In this study, ‘kinnow’ mandarin (Citrus nobilis X Citrus deliciosa) biomass was studied for its flavonoid profile. For this, dried and pulverized peels were subjected to green and sustainable extraction techniques, namely, supercritical fluid extraction carried out under conditions pressure: 330 bar, temperature: 40 ̊ C and co-solvent: 10% ethanol. The obtained extract was observed to contain 47.3±1.06 mg/ml rutin equivalents as total flavonoids. Mass spectral analysis revealed the prevalence of polymethoxyflavones (PMFs), chiefly tangeretin and nobiletin. Furthermore, the antioxidant potential was analyzed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, which was estimated to be at an IC₅₀ of 0.55μg/ml. The pre-systemic metabolism of flavonoids limits their functionality, as was observed in this study through in vitro gastrointestinal studies where nearly 50.0% of the flavonoids were degraded within 2 hours of gastric exposure. We proposed nanoencapsulation as a means to overcome this problem, and flavonoids-laden polylactic-co-glycolic acid (PLGA) nano encapsulates were bioengineered using solvent evaporation method, and these were furnished to a particle size between 200-250nm, which exhibited protection of flavonoids in the gastric environment, allowing only 20% to be released in 2h. A further step involved impregnating the nano encapsulates within alginate hydrogels which were fabricated by ionic cross-linking, which would act as delivery vehicles within the gastrointestinal (GI) tract. As a result, 100% protection was achieved from the pre-systemic release of bioflavonoids. These alginate hydrogels had key significant features, i.e., less porosity of nearly 20.0%, and Cryo-SEM (Cryo-scanning electron microscopy) images of the composite corroborate the packing ability of the alginate hydrogel. As a result of this work, it is concluded that the waste can be used to develop functional biomaterials while retaining the functionality of the bioactive itself.

Keywords: bioflavonoids, gastrointestinal, hydrogels, mandarins

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Authors: Delphine Morales, Florian Lombart, Agathe Truchot, Pauline Maire, Pascale Vigneron, Antoine Galmiche, Catherine Lok, Muriel Vayssade

Abstract:

Targeted therapy molecules are used as a first-line treatment for metastatic melanoma with B-Raf mutation. Nevertheless, these molecules can cause side effects to patients and are efficient on 50 to 60 % of them. Indeed, melanoma cell sensitivity to targeted therapy molecules is dependent on tumor microenvironment (cell-cell and cell-extracellular matrix interactions). To better unravel factors modulating cell sensitivity to B-Raf inhibitor, we have developed and compared several melanoma models: from metastatic melanoma cells cultured as monolayer (2D) to a co-culture in a 3D dermal equivalent. Cell response was studied in different melanoma cell lines such as SK-MEL-28 (mutant B-Raf (V600E), sensitive to Vemurafenib), SK-MEL-3 (mutant B-Raf (V600E), resistant to Vemurafenib) and a primary culture of dermal human fibroblasts (HDFn). Assays have initially been performed in a monolayer cell culture (2D), then a second time on a 3D dermal equivalent (dermal human fibroblasts embedded in a collagen gel). All cell lines were treated with Vemurafenib (a B-Raf inhibitor) for 48 hours at various concentrations. Cell sensitivity to treatment was assessed under various aspects: Cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK signaling pathway analysis (Western-Blotting), Apoptosis (TUNEL), Cytokine release (IL-6, IL-1α, HGF, TGF-β, TNF-α) upon Vemurafenib treatment (ELISA) and histology for 3D models. In 2D configuration, the inhibitory effect of Vemurafenib on cell proliferation was confirmed on SK-MEL-28 cells (IC50=0.5 µM), and not on the SK-MEL-3 cell line. No apoptotic signal was detected in SK-MEL-28-treated cells, suggesting a cytostatic effect of the Vemurafenib rather than a cytotoxic one. The inhibition of SK-MEL-28 cell proliferation upon treatment was correlated with a strong expression decrease of phosphorylated proteins involved in the MAPK pathway (ERK, MEK, and AKT/PKB). Vemurafenib (from 5 µM to 10 µM) also slowed down HDFn proliferation, whatever cell culture configuration (monolayer or 3D dermal equivalent). SK-MEL-28 cells cultured in the dermal equivalent were still sensitive to high Vemurafenib concentrations. To better characterize all cell population impacts (melanoma cells, dermal fibroblasts) on Vemurafenib efficacy, cytokine release is being studied in 2D and 3D models. We have successfully developed and validated a relevant 3D model, mimicking cutaneous metastatic melanoma and tumor microenvironment. This 3D melanoma model will become more complex by adding a third cell population, keratinocytes, allowing us to characterize the epidermis influence on the melanoma cell sensitivity to Vemurafenib. In the long run, the establishment of more relevant 3D melanoma models with patients’ cells might be useful for personalized therapy development. The authors would like to thank the Picardie region and the European Regional Development Fund (ERDF) 2014/2020 for the funding of this work and Oise committee of "La ligue contre le cancer".

Keywords: 3D human skin model, melanoma, tissue engineering, vemurafenib efficiency

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Authors: Katarína Koňariková, Georgios A. Perdikaris, Lucia Andrezálová, Zdeňka Ďuračková, Lucia Laubertová, Helena Gbelcová, Ingrid Žitňanová

Abstract:

Introduction: The continuous demand for new anti-cancer drugs has stimulated chemotherapeutic research based on the use of essential metalloelements with the aim to develop potential drugs with lower toxicity and higher antiproliferative activity against tumors. Copper(II) and its complexes play an important role as suitable species for antiproliferative tests. Objectives: The central objective of the current study was to investigate the potential in vitro anti-proliferative effects of N-salicylidene-L-glutamato copper (II) complexes and molecular mechanism of apoptosis induced by tested complexes. In our project we tested N-salicylidene-L-glutamato copper (II) complexes ZK1 - [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK0 - ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O); MK1 - [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol); MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] at concentration range 0.001-100 µmol/L against human colon carcinoma cell line HT-29 and human breast carcinoma cell line MCF-7. Methods: Viability was assessed by direct counting of 0.4% trypan blue dye-excluding cells after 24, 48 and 72 hour cultivations with or without copper complex and by MTT assay. To analyze the type of cell death and its mechanism induced by our copper complex we used different methods. To distinguish apoptosis from necrosis we used electrophoretic analysis, to study the activity of caspases 8 and 9 – luminometric analysis and caspase activity 3 colorimetric assay. Results: The observed anti-proliferative effect of the copper complexes appeared to be dose-, time- and cell line- dependent. Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) than to ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)). Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex than human breast carcinoma cells MCF-7. IC50 decreased with time of incubation (24, 48 and 72h) for HT-29, but increased for MCF-7. By electrophoresis we found apoptotic cell death induced by our copper complexes in HT-29 at concentrations 1, 10, 50 and 100 µmol/L after 48h (ZK1) and 72h (MK0, MK1) and in MCF-7 we did not find apoptosis. We also studied molecular mechanism of apoptosis in HT-29 induced by copper complexes. We found active caspase 9 in HT-29 after ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)) influence and active caspase 8 after MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) influence. Conclusion: Our copper complexes showed cytotoxic activities against human colon carcinoma cells HT-29 and breast cancer cell line MCF-7 in vitro. Apoptosis was activated by mitochondrial pathway (intrinsic pathway) in case of ZK1 [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK1 [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol) and MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] copper complexes and by death receptors (extrinsic pathway) in case of MK0 [Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O copper complex in HT-29.

Keywords: apoptosis, copper complex, cancer, carcinoma cell line

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