- Email: [email protected]
Nucleolin and its position in cancer therapy
New Biotechnology · Volume 25S · September 2009
the reasons that account for the noncytotoxic ability of HP-RNase is its inhibition by the proteic ribonuclease inhibitor present in the cytosol of mammalian cells. We have reasoned that directing this enzyme to a cellular compartment devoid of this inhibitor would endow it with the desired property. Thus, we have constructed an HP-RNase variant, named PE5, which presents a nonclassical nuclear localization sequence (NLS) responsible for its import to the nucleus. Accordingly, unlike ONC, this variant degrades mainly nuclear RNA. Its cytotoxic properties have been tested on different tumor cell lines being one of the most sensible ovarian cell line (NCI-ADR/RES) that presents multiple drug resistance phenotype at least in part associated with the overexpression of P-glycoprotein (P-gp). The cytotoxic mechanism of PE5 was investigated on this cell line. The results showed that cellular death is produced by apoptosis. The apoptotic process was evidenced by the visualization of cell morphology using confocal microscopy, cell staining with annexin V-Alexa Fluor 488 and propidium iodide followed by FACS and by assaying caspase-3, -8 and -9 activation. Unlike ONC, the cytotoxic effect of PE5 is produced mainly in phase G0/G1 of the cell cycle. Selectivity studies carried out comparing the cytoxicity of PE5 on tumor and on normal human cell lines show similar results to those found for ONC. Finally, PE5 presents synergy with doxorubicin on NCI-ADR-RES cell line. This effect is explained by a specific inhibition of P-gp expression. Altogether the results suggest that PE5 can be an antitumor drug candidate. Its human origin may endow it with an increased immunological tolerance and reduced renal toxicity when compared with ONC. doi:10.1016/j.nbt.2009.06.023
1.1.19 Synthesis and antimicrobial activities of some novel benzimidazole—hydrazone derivatives M.G. Ozarda
ABSTRACTS
on same structure to investigate their probable antimicrobial activity on several microorganisms. Some of the compounds showed antimicrobial activity to different extents and compounds 3 and 8 found to be the most active derivatives in the series. doi:10.1016/j.nbt.2009.06.024
1.1.20 Anti-inflammatory effect of chitosan oligomers H. Spindola 1,∗ , J. Fernandes 2 , V. De Sousa 3 , F. Tavaria 2 , M. Pintado 2 , X. Malcata 2 , J.E. Carvalho 3 1
CPQBA/FOP - UNICAMP, Campinas, Brazil CBQF - Escola Superior de Biotecnologia, Brazil 3 CPQBA - UNICAMP, Brazil 2
Chitosan has been used as source of potential bioactive material in the past few decades. However, in the biomedical field, chitooligosaccharides (COS) are more applicable due to their water solubility and higher absorption profiles at intestinal level — quickly getting into the blood flow and having systemic biological effects in the organism. To explore the anti-inflammatory activities of COS, we tested two COS mixtures (obtained via enzymatic, with molecular weight <3 and <5 kDa) at several concentrations, by carrageenan-induced paw edema methodology, in mice. The inflammatory response was quantified by increase in paw size (edema) which is maximal around five-hour postcarrageenan injection, and is modulated by inhibitors of specific molecules within the inflammatory cascade. COS showed anti-inflammatory action, which increased with the molecular weight. COS <5 kDa even showed higher effect than the control, indomethacin, after two hours, with no harmful secondary effects. Our data suggest that COS possesses anti-inflammatory effect, dose and MW-dependent. doi:10.1016/j.nbt.2009.06.025
Pharmacy Faculty, Eskisehir, Turkey
1.1.21 The development of resistance to current antibacterial therapy continues to drive the search for more effective agents. In addition, primary and opportunistic fungal infections continue to increase the number of immunocompromised patients, those suffering from such as AIDS or cancer or who have undergone organ transplantation. It is well known that benzimidazole derivatives are very important compounds in medicinal and pharmaceutical field. There are several studies including biological activities of benzimidazolerelated compounds. Benzimidazoles exhibit antimicrobial, antitubercular, anticancer, anthelmintic, antiallergic, antioxidant, anticonvulsant and analgesic activities. It is also well known that hydrazones, containing an azometine —NHN CH— group, constitute an important class of compounds for new drug development. Hydrazone derivatives possess antimicrobial, anticonvulsant, analgesic, anti-inflammatory, antiplatelet, antitubercular and antitumoral activities. Prompted above observations we synthesized 14 novel compounds including benzimidazole and hydrazone pharmacophores
Nucleolin and its position in cancer therapy M. Tabarzad ∗ , F. Hosseini Shirazi Pharmaceutical Biotechnology Department, Tehran, Islamic Republic of Iran
Extracellular signalling molecules such as growth factors and cytokines bind to plasma membrane receptors that activate their own kinases and/or recruit adapter proteins. These events on the plasma membrane have been regarded as the onset of signalling. Subsequent to receptor binding of ligands, the ligand—receptor complexes are internalized and delivered to specific vesicular compartments, leading to desensitization. While the ligands are often degraded, the receptors themselves are either degraded or recycled back to the cell surface. Mounting evidence indicates that nuclear targeting by extracellular signalling molecules plays an indispensable role in their biological activities. These signalling pathways could be the target of pharmaceutical agents and show different role in their pharmacological effects.
www.elsevier.com/locate/nbt S9
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
One of these signalling receptors, regard to increasing investigations, which could have important function in proliferation at cancerous cells, is nucleolin. Nucleolin is a ubiquitous eukaryotic protein conserved through yeast to human. Nucleolin, with a unique multiple domain structure, plays a fascinating and fundamental role in linking nucleolar activities to cell proliferation and mitotic dynamics. The nucleolin polypeptide consists of a negatively charged NH2-terminal domain, an RNA-binding domain and a COOHterminal domain rich in RGG motifs. The main functions of nucleolin relate to rRNA maturation and ribosome assembly. Although nucleolin was originally described as a nuclear and cytoplasmic protein, several studies show that it can also be expressed at the cell surface. Recent results also ascribe additional functions to nucleolin as a shuttle protein between the cytoplasm and the nucleus, and between the cell surface and the nucleus. The expression of nucleolin at the cell surface seems to correlate with growth and metabolic activity of cells. Different natural agents with anticancer activity, like achran sulphate, show specific affinity for binding to cell surface nucleolin and respect on its role in cell growth and activity, therefore many studies made to investigate its modulating agents as effective anticancer means. Among these efforts, discovery of an anticancer aptamer could be considered as attractive result which the mechanism of its antiproliferative effects in cancer cells seems to involve initial binding to cell surface nucleolin and internalization, leading to an inhibition of DNA replication. In this review, we study new insights of nucleolin role and its modulation agents in different type of cancers.
sue samples from wound site of each animal were removed for histopathological analysis and for total collagen determination. The anti-ulcerative assay was done in healthy adult (250—300 g) male Wistar rats (R. norvegicus), n = 5/group, using carbenoxolone as a positive control. Compounds were administered orally according to their body weight (1000 mg/kg). After 30 min, each animal received 1 mL of absolute ethanol orally. After one hour, animals were sacrificed by cervical displacement and their stomachs were removed, opened along the greater curvature and the ulcerative lesion index was determined. Ear oedema was used to evaluate the anti-inflammatory effect of chitosans in Swiss adult mice (Mus musculus) weighing approximately 30—40 g. Croton oil was used as the inflammatory agent and dexamethazone as positive control. The oedema was measured by subtracting the weight of the ear receiving only acetone (vehicle) by that receiving the irritating agent. The oedema inhibition (%) was determined by the formula: [(wC − wT )/wC ] × 100, where wC was the ear weight in the croton oil group and wT was the ear weight in the treatment groups. The anti-proliferative activity of chitosan was evaluated in vitro against eight human tumor cell lines. The Total Growth Inhibition (cytostatic activity) was determined for each compound. The in vitro cytotoxic activity of chitosan was found to be minimal, whereas it showed wound healing ability. The ear oedema assay showed no differences among the croton oil group and chitosan groups, demonstrating no anti-inflammatory activity. High molecular weight chitosan seems to render gut protection, but the medium molecular weight is the best anti-ulcerative compound. doi:10.1016/j.nbt.2009.06.027
doi:10.1016/j.nbt.2009.06.026 1.1.23 1.1.22 Wound healing, anti-ulcerogenic, anti-inflammatory and anti-proliferative properties of chitosan F. Tavaria 1,∗ , M. Jorge 2 , G. Marchetti 2 , V. Souza 2 , A.L. Ruíz 2 , X. Malcata 1 , M. Pintado 1 , J. Carvalho 2
Anti-inflammatory effect of naringin and naringenin on TNF-␣ secretion in cultured cortical astrocytes after stimulation with LPS M. Ribeiro ∗ , A. Barateiro, H. Vila-Real, A. Fernandes, D. Brites Faculdade Farmácia, University of Lisbon, Lisbon, Portugal
1
Universidade Católica Portuguesa, Porto, Portugal 2 Brazil
Chitosan, is one of the most abundant, renewable, nontoxic and biodegradable carbohydrate polymers, and is largely available in the exoskeletons of shellfish and insects. It has recently attracted great attention in the pharmaceutical, biomedical and food arenas, due to its favourable physico-chemical and biological properties. In this research effort, we have assessed wound healing, antiulcerogenic and anti-inflammatory properties of chitosan in vivo, and the anti-proliferative activity in vitro. We used three chitosans differing in their molecular weight and degree of deacetylation. The wound healing study was performed using healthy adult (250—300 g) male Wistar rats (Rattus norvegicus), n = 5/group. After thiopental anaesthesia, excision wounds sized 1.5 cm2 in average were made. Wounds were treated topically for ten days by applying 200 L/wound of each sample (0.2 mg/mL) and protected by a wound curative. On day 11, animals were sacrificed and tisS10
www.elsevier.com/locate/nbt
Glycosides of polyphenolic compounds have been described for their antioxidant, anti-inflammatory and neuroprotective activities. Deglycosylated naringin (naringenin) was previously shown to have more efficacies in the treatment of inflammation in a model of -carrageenan induced paw in mice. Lipopolysaccharide (LPS) induces the release of TNF-␣ in nerve cells such as astrocytes and indomethacin was pointed to abrogate this effect. Thus, in this study we aimed to: (i) determine the efficacy of the anti-inflammatory properties of both naringin and naringenin in LPS-stimulated astrocytes and (ii) to establish the efficiency of each compound relatively to that of indomethacin. Primary cultures of rat cortical astrocytes cultivated for ten days in vitro were incubated for 4, 8 and 24 hours with 10 ng/ml LPS, 0.1 ng/ml indomethacin, 3.2 g/ml naringin or 10 g/ml naringenin, at 37◦ C. In sister cultures cells were incubated with LPS + naringin, LPS + indomethacin or LPS + naringenin. Cytokine release was assessed by ELISA.
the reasons that account for the noncytotoxic ability of HP-RNase is its inhibition by the proteic ribonuclease inhibitor present in the cytosol of mammalian cells. We have reasoned that directing this enzyme to a cellular compartment devoid of this inhibitor would endow it with the desired property. Thus, we have constructed an HP-RNase variant, named PE5, which presents a nonclassical nuclear localization sequence (NLS) responsible for its import to the nucleus. Accordingly, unlike ONC, this variant degrades mainly nuclear RNA. Its cytotoxic properties have been tested on different tumor cell lines being one of the most sensible ovarian cell line (NCI-ADR/RES) that presents multiple drug resistance phenotype at least in part associated with the overexpression of P-glycoprotein (P-gp). The cytotoxic mechanism of PE5 was investigated on this cell line. The results showed that cellular death is produced by apoptosis. The apoptotic process was evidenced by the visualization of cell morphology using confocal microscopy, cell staining with annexin V-Alexa Fluor 488 and propidium iodide followed by FACS and by assaying caspase-3, -8 and -9 activation. Unlike ONC, the cytotoxic effect of PE5 is produced mainly in phase G0/G1 of the cell cycle. Selectivity studies carried out comparing the cytoxicity of PE5 on tumor and on normal human cell lines show similar results to those found for ONC. Finally, PE5 presents synergy with doxorubicin on NCI-ADR-RES cell line. This effect is explained by a specific inhibition of P-gp expression. Altogether the results suggest that PE5 can be an antitumor drug candidate. Its human origin may endow it with an increased immunological tolerance and reduced renal toxicity when compared with ONC. doi:10.1016/j.nbt.2009.06.023
1.1.19 Synthesis and antimicrobial activities of some novel benzimidazole—hydrazone derivatives M.G. Ozarda
ABSTRACTS
on same structure to investigate their probable antimicrobial activity on several microorganisms. Some of the compounds showed antimicrobial activity to different extents and compounds 3 and 8 found to be the most active derivatives in the series. doi:10.1016/j.nbt.2009.06.024
1.1.20 Anti-inflammatory effect of chitosan oligomers H. Spindola 1,∗ , J. Fernandes 2 , V. De Sousa 3 , F. Tavaria 2 , M. Pintado 2 , X. Malcata 2 , J.E. Carvalho 3 1
CPQBA/FOP - UNICAMP, Campinas, Brazil CBQF - Escola Superior de Biotecnologia, Brazil 3 CPQBA - UNICAMP, Brazil 2
Chitosan has been used as source of potential bioactive material in the past few decades. However, in the biomedical field, chitooligosaccharides (COS) are more applicable due to their water solubility and higher absorption profiles at intestinal level — quickly getting into the blood flow and having systemic biological effects in the organism. To explore the anti-inflammatory activities of COS, we tested two COS mixtures (obtained via enzymatic, with molecular weight <3 and <5 kDa) at several concentrations, by carrageenan-induced paw edema methodology, in mice. The inflammatory response was quantified by increase in paw size (edema) which is maximal around five-hour postcarrageenan injection, and is modulated by inhibitors of specific molecules within the inflammatory cascade. COS showed anti-inflammatory action, which increased with the molecular weight. COS <5 kDa even showed higher effect than the control, indomethacin, after two hours, with no harmful secondary effects. Our data suggest that COS possesses anti-inflammatory effect, dose and MW-dependent. doi:10.1016/j.nbt.2009.06.025
Pharmacy Faculty, Eskisehir, Turkey
1.1.21 The development of resistance to current antibacterial therapy continues to drive the search for more effective agents. In addition, primary and opportunistic fungal infections continue to increase the number of immunocompromised patients, those suffering from such as AIDS or cancer or who have undergone organ transplantation. It is well known that benzimidazole derivatives are very important compounds in medicinal and pharmaceutical field. There are several studies including biological activities of benzimidazolerelated compounds. Benzimidazoles exhibit antimicrobial, antitubercular, anticancer, anthelmintic, antiallergic, antioxidant, anticonvulsant and analgesic activities. It is also well known that hydrazones, containing an azometine —NHN CH— group, constitute an important class of compounds for new drug development. Hydrazone derivatives possess antimicrobial, anticonvulsant, analgesic, anti-inflammatory, antiplatelet, antitubercular and antitumoral activities. Prompted above observations we synthesized 14 novel compounds including benzimidazole and hydrazone pharmacophores
Nucleolin and its position in cancer therapy M. Tabarzad ∗ , F. Hosseini Shirazi Pharmaceutical Biotechnology Department, Tehran, Islamic Republic of Iran
Extracellular signalling molecules such as growth factors and cytokines bind to plasma membrane receptors that activate their own kinases and/or recruit adapter proteins. These events on the plasma membrane have been regarded as the onset of signalling. Subsequent to receptor binding of ligands, the ligand—receptor complexes are internalized and delivered to specific vesicular compartments, leading to desensitization. While the ligands are often degraded, the receptors themselves are either degraded or recycled back to the cell surface. Mounting evidence indicates that nuclear targeting by extracellular signalling molecules plays an indispensable role in their biological activities. These signalling pathways could be the target of pharmaceutical agents and show different role in their pharmacological effects.
www.elsevier.com/locate/nbt S9
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
One of these signalling receptors, regard to increasing investigations, which could have important function in proliferation at cancerous cells, is nucleolin. Nucleolin is a ubiquitous eukaryotic protein conserved through yeast to human. Nucleolin, with a unique multiple domain structure, plays a fascinating and fundamental role in linking nucleolar activities to cell proliferation and mitotic dynamics. The nucleolin polypeptide consists of a negatively charged NH2-terminal domain, an RNA-binding domain and a COOHterminal domain rich in RGG motifs. The main functions of nucleolin relate to rRNA maturation and ribosome assembly. Although nucleolin was originally described as a nuclear and cytoplasmic protein, several studies show that it can also be expressed at the cell surface. Recent results also ascribe additional functions to nucleolin as a shuttle protein between the cytoplasm and the nucleus, and between the cell surface and the nucleus. The expression of nucleolin at the cell surface seems to correlate with growth and metabolic activity of cells. Different natural agents with anticancer activity, like achran sulphate, show specific affinity for binding to cell surface nucleolin and respect on its role in cell growth and activity, therefore many studies made to investigate its modulating agents as effective anticancer means. Among these efforts, discovery of an anticancer aptamer could be considered as attractive result which the mechanism of its antiproliferative effects in cancer cells seems to involve initial binding to cell surface nucleolin and internalization, leading to an inhibition of DNA replication. In this review, we study new insights of nucleolin role and its modulation agents in different type of cancers.
sue samples from wound site of each animal were removed for histopathological analysis and for total collagen determination. The anti-ulcerative assay was done in healthy adult (250—300 g) male Wistar rats (R. norvegicus), n = 5/group, using carbenoxolone as a positive control. Compounds were administered orally according to their body weight (1000 mg/kg). After 30 min, each animal received 1 mL of absolute ethanol orally. After one hour, animals were sacrificed by cervical displacement and their stomachs were removed, opened along the greater curvature and the ulcerative lesion index was determined. Ear oedema was used to evaluate the anti-inflammatory effect of chitosans in Swiss adult mice (Mus musculus) weighing approximately 30—40 g. Croton oil was used as the inflammatory agent and dexamethazone as positive control. The oedema was measured by subtracting the weight of the ear receiving only acetone (vehicle) by that receiving the irritating agent. The oedema inhibition (%) was determined by the formula: [(wC − wT )/wC ] × 100, where wC was the ear weight in the croton oil group and wT was the ear weight in the treatment groups. The anti-proliferative activity of chitosan was evaluated in vitro against eight human tumor cell lines. The Total Growth Inhibition (cytostatic activity) was determined for each compound. The in vitro cytotoxic activity of chitosan was found to be minimal, whereas it showed wound healing ability. The ear oedema assay showed no differences among the croton oil group and chitosan groups, demonstrating no anti-inflammatory activity. High molecular weight chitosan seems to render gut protection, but the medium molecular weight is the best anti-ulcerative compound. doi:10.1016/j.nbt.2009.06.027
doi:10.1016/j.nbt.2009.06.026 1.1.23 1.1.22 Wound healing, anti-ulcerogenic, anti-inflammatory and anti-proliferative properties of chitosan F. Tavaria 1,∗ , M. Jorge 2 , G. Marchetti 2 , V. Souza 2 , A.L. Ruíz 2 , X. Malcata 1 , M. Pintado 1 , J. Carvalho 2
Anti-inflammatory effect of naringin and naringenin on TNF-␣ secretion in cultured cortical astrocytes after stimulation with LPS M. Ribeiro ∗ , A. Barateiro, H. Vila-Real, A. Fernandes, D. Brites Faculdade Farmácia, University of Lisbon, Lisbon, Portugal
1
Universidade Católica Portuguesa, Porto, Portugal 2 Brazil
Chitosan, is one of the most abundant, renewable, nontoxic and biodegradable carbohydrate polymers, and is largely available in the exoskeletons of shellfish and insects. It has recently attracted great attention in the pharmaceutical, biomedical and food arenas, due to its favourable physico-chemical and biological properties. In this research effort, we have assessed wound healing, antiulcerogenic and anti-inflammatory properties of chitosan in vivo, and the anti-proliferative activity in vitro. We used three chitosans differing in their molecular weight and degree of deacetylation. The wound healing study was performed using healthy adult (250—300 g) male Wistar rats (Rattus norvegicus), n = 5/group. After thiopental anaesthesia, excision wounds sized 1.5 cm2 in average were made. Wounds were treated topically for ten days by applying 200 L/wound of each sample (0.2 mg/mL) and protected by a wound curative. On day 11, animals were sacrificed and tisS10
www.elsevier.com/locate/nbt
Glycosides of polyphenolic compounds have been described for their antioxidant, anti-inflammatory and neuroprotective activities. Deglycosylated naringin (naringenin) was previously shown to have more efficacies in the treatment of inflammation in a model of -carrageenan induced paw in mice. Lipopolysaccharide (LPS) induces the release of TNF-␣ in nerve cells such as astrocytes and indomethacin was pointed to abrogate this effect. Thus, in this study we aimed to: (i) determine the efficacy of the anti-inflammatory properties of both naringin and naringenin in LPS-stimulated astrocytes and (ii) to establish the efficiency of each compound relatively to that of indomethacin. Primary cultures of rat cortical astrocytes cultivated for ten days in vitro were incubated for 4, 8 and 24 hours with 10 ng/ml LPS, 0.1 ng/ml indomethacin, 3.2 g/ml naringin or 10 g/ml naringenin, at 37◦ C. In sister cultures cells were incubated with LPS + naringin, LPS + indomethacin or LPS + naringenin. Cytokine release was assessed by ELISA.