Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S441

Reference

Kanayama, N., et al., 2006. Nucleic Acids Res. 34, e10.

doi:10.1016/j.jbiotec.2010.09.628

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Magnetic force-based tissue engineering of skeletal muscle

Yasunori Yamamoto 1,∗, Akira Ito 1, Yoshinori Kawabe 1, HideakiFujita 2, Eiji Nagamori 2, Masamichi Kamihira 1

1 Kyushu University, Japan2 Toyota Central R&D Labs. Inc., JapanKeywords: Skeletal muscle; Tissue engineering; MCL; C2C12myoblast cells

Skeletal muscle tissue engineering will be useful for regen-erative medicine, drug screening and bio-actuator. It is requiredto fabricate bio-mimic and functional skeletal muscle tissuesto achieve these purposes. To construct artificial tissues, mag-netite cationic liposomes (MCLs), in which magnetite nanoparticleswere encapsulated by cationic liposomes, were used for mag-netic labelling of cells, and the magnetically labelled cells wereaccumulated and self-organized by applying magnetic force. Themethodology has been designated the magnetic force-based tissueengineering (Mag-TE) technique. In the present study, we fabri-cated highly cell dense and oriented muscular tissues using theMag-TE technique. A silicone plug was positioned at the center ofa well whose surface was coated with a neutrally charged hydro-gel, and magnetically labeled C2C12 myoblast cells were seededinto the well. When a magnet was placed under the well, mag-netically labeled cells were accumulated by magnetic force andmultilayered cell sheets formed around the plug. Thereafter, the cellsheets drastically shrank and formed a ring-like assembly aroundthe plug. Then, the cellular ring was hooked around two pins andcultured for 7 d in a differentiation medium. Histological analy-sis revealed that a highly cell dense and oriented muscular tissuewas successfully constructed. Differentiated multinucleated cellswere observed within the muscle tissue constructs. Biochemicaland mechanical evaluation of the artificial muscle tissues were per-formed. Western blot analysis and creatine kinase activity assayrevealed that muscle specific proteins increased for at least 17 days.With the stimulation by electric pulse, the artificial muscle tissuecontracted in response to the change in voltages, pulse widths andfrequencies. These results indicate that this procedure can providea novel strategy for skeletal muscle tissue engineering.

doi:10.1016/j.jbiotec.2010.09.629

[P-M.42]

effect of celecoxib on P-ERK, COX-2, PGE2 and VEGF expressionin oral cancer in presence and absence of nicotine

Mona Salimi 1,∗, Masoumeh Esfahani 2, Nooshin Rastkari 3, BitaSedaghati 1, Mohammad Hossein Ghahremani 4

1 Research & Development Department,Pasteur Institute ofIran,Tehran, Islamic Republic of Iran2 Payame Noor University,Tehran, Islamic Republic of Iran3 Center for Environmental Research (CER), Tehran University of Med-ical SciencesTehran University of Medical Sciences,Tehran, IslamicRepublic of Iran4 Toxicology & Pharmacology Department Pharmacy Faculty, TehranUniversity of Medical Sciences,Tehran, Islamic Republic of IranKeywords: Cancer; Celecoxib; Nicotine; Oral

Introduction: A new concept indicates that chronic inflamma-tion plays a crucial role in promoting of certain cancers such as oralcancer.Oral cancer is a common neoplasm worldwide, particularlyin developing countries.Yet oral cancer is an excellent candidatefor treatment a chemopreventive strategy. Prostaglandines(PGs)are known to play important roles in the proliferation of varioustypes of cancer.PGs are induced by the action of cyclooxyge-nase(COX) enzymes. Over-expression of ERK-1/2(Extra cellularsignal–regulated kinases) and VEGF are also found in most tumors(Okada et al., 2000; Minter et al., 2003; Shin et al., 2004). In ourprevious study, the role of nicotine on COX-2, P-ERK, PGE2 andVEGF expression were also investigated. Considering these dataand knowing that the use of COX-2 inhibitors can prevent cancerprogression, it was important for us to understand the signalingpathway to design preventive strategies for this disease in smokersand non-smokers.

Methods: Cytotoxicity was investigated by using MTT assay andwestern blotting was used to assess COX-2 and P-ERK expres-sion. VEGF and PGE2 were measured by Elisa Kit. Celecoxib(1,10,100 �g/mL)was added one hour before nicotine stimula-tion.Nicotine(50,200 �g/mL)was added to search for the possiblemechanisms of nicotine-induced cells.

Results: Results showed that celecoxib exerted cytotoxicitydose-dependently on the BHY cells. (Cytotoxicity at 1,10,100 �g/mLof celecoxib are 5,7,56%).Our studies demonstrated that celecoxibin presence and absence of nicotine couldn’t activate the phospho-rylation of ERK in comparison with control. VEGF and PGE2 levelwas decreased in celecoxib (100 �g/mL) treated cells. In nicotine-stimulated cells(200,50 �g/mL), celecoxib 1, 10,100 �g/mL coulddecrease PGE2 expression.VEGF expression was insignificantlydecreased (p<0.05) by 1 �g/mL celecoxib in these cells, howeverincreased by 10,100 �g/mL celecoxib in comparison with nicotine-stimulated cells.

Discussion: In conclusion, celecoxib can have cytotoxicity effectsin squamous oral cancer and can change the effects of nicotinedose-dependently. This,together with its anti-inflammatory effectprovides a rational for potential use of celecoxib in oral cancer forsmokers and non-smokers individuals.

References

Okada, F., Kawaguchi, T., Habelhah, H., Kobayashi, T., Tazawa, H., Takeichi, N., Kita-gawa, T., Hosokawa, M., 2000. Conversion of human colonic adenoma cellsto adenocarcinoma cells through inflammation in nude mice. Lab Invest 80,1617–1628.

Minter, H.A., Eveson, J.W., Huntley, S., Elder, D.J.E., Hague, A., 2003. The cyclooxy-genase 2-selective inhibitor NS398 inhibits proliferation of oral carcinoma celllinesby mechanisms dependent and independent of reduced prostaglandin E2synthesis. Clin Can Res 9, 1885–1897.

Shin, V.Y., Wu, W.K.K., Ye, Y.N., So, W.H.L., Koo, M.W.L., Liu, E.S.L., Luo, J.C., Cho,C.H., 2004. Nicotine promotes gastric tumor growth and neovascularization by

S442 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

activating extracellular signal-regulated kinase and cyclooxygenase-2. Carcino-genesis 25 (12), 2487–2495.

doi:10.1016/j.jbiotec.2010.09.630

[P-M.43]

Nicotine effect on P-ERK, COX-2, PGE2 and VEGF expression inoral squamous cancer

Minoo Hamraz 1, Mona Salimi 1, Masoumeh Esfahani 2, BitaSedaghati 1, Hamid Reza Aslani 3, Azadeh Bazarchi 1,∗

1 Research & Development Department, Pasteur Institute ofIran,Tehran, Islamic Republic of Iran2 Payame Noor University,Tehran, Islamic Republic of Iran3 Toxicology & Pharmacology Department, Pharmacy Faculty, TehranUniversity of Medical Science,Tehran, Islamic Republic of IranKeywords: nicotine; VEGF; COX; PGE2

Introduction: Cigarette smoking is a major public health issueand is associated with Cancer.There are numerous compounds incigarette smoke making it difficult to study their adverse action inbiological system. Recent researches have focused on the deleteri-ous effects of cigarette smoke particularly on the oral cancer.Studieshave shown that nicotine can induce cyclooxygenase-2(COX-2)expression and increase the release of prostaglandine E2 andVEGF in gastric cancer cells.COX-2 is not only a marker of inflam-mation,but has also been reported to have significant relevanceto carcinogenesis tissue.Over-expression of ERK-1/2(extra cellu-lar signal–regulated kinases)is also found in most tumors andis involved in nicotine-induced cell proliferation (Ho and Chang,2006; Shin et al., 2005; Shin et al., 2004).To elucidate the possiblesignaling pathway of nicotine in oral cancer,we investigated theeffect of nicotine on expression of COX-2,P-ERK,PGE2,VEGF in oralsquamous cell carcinoma(BHY).

Methods: BHY cell was obtained from DSMZ bank and grown in 6well plates in the absence and presence of nicotine (50, 200 �g/ml)for 5 h. Western blotting was used to assess COX-2 and P-ERKexpression.VEGF and PGE2 was measured by Elisa Kit.Cell viabilitywas measured by MTT assay in 96 well plates.

Results: Results showed that nicotine exerted no cytotoxicityeffects on the cells(%viability at 50 �g/ml = 94%, 200 �g/ml = 100%)in comparison with control cells.Our findings demonsrated thatnicotine couldn’t activate the phosphorylation of ERK in compari-son with control cells.To evaluate the correlation between nicotineand COX-2, COX-2 expression and PGE2 secretion were determinedin nicotine-treated cells.Nicotine(200,50 �g/ml)could decrease thePGE2 concentration dose-dependently by∼ 64 and70%, respec-tively.we measured the level of VEGF in BHY cells.Results showedthat high dose of nicotine decreased VEGF expression insignif-icantly in comparison with control cells, but low dose couldsignificantly decrease it(p<0.05)

Discussion: This in vitro study signifies the involvement of P-ERK/COX-2/PGE2/VEGF expression in oral cancer.In conclusion,ourfindings suggest probable mechanism of action of nicotine whichcan be targeted for chemopreventive agents.

References

Ho, Y.C., Chang, Y.C., 2006. Regulation of nicotine-induced cyclooxygenase-2 proteinexpression in human gingival fibroblasts. Acta Pharm Sinica 27 (4), 409–413.

Shin, V.Y., Wu, W.K.K., Chu, K.M., Wong, H.P.S., Lam, E.K.Y., Tai, E.K.K., Koo, M.W.L.,Cho, C.H., 2005. Nicotine induces cyclooxygenase-2 and vascular endothelialgrowth factor receptor-2 in association with tumor-associated invasion andangiogenesis in gastric cancer. Mol Cancer Res 3 (11), 607–615.

Shin, V.Y., Wu, W.K.K., Ye, Y.N., So, W.H.L., Koo, M.W.L., Liu, E.S.L., Luo, J.C., Cho,C.H., 2004. Nicotine promotes gastric tumor growth and neovascularization by

activating extracellular signal-regulated kinase and cyclooxygenase-2. Carcino-genesis 25 (12), 2487–2495.

doi:10.1016/j.jbiotec.2010.09.631

[P-M.44]

Site-directed mutagenesis of D-amino acid oxidase from yeastTrigonopsis variabilis

N.V. Cherskova 1,2,∗, S.V. Khoronenkova 2, M.A. Panteleev 3, V.I.Tishkov 2,3

1 A.N.Bach Institute of Biochemistry of the Russian Academy of Sci-ences, Russian Federation2 Innovations and High Technologies MSU Ltd, Russian Federation3 Chemical Enzymology Department, Chemistry Faculty, M.V.Lomonosov Moscow State University, Russian FederationKeywords: D-amino acid oxidase; Thermal stability; Site-directedmutagenesis

D-amino acid oxidase is a FAD-containing enzyme catalyzing D-amino acids oxidation to corresponding �-keto acids. Being widelyoccurred in nature, DAAO plays important role in metabolism ofvarious organisms, including human, and is highly essential forpractical use as well. Wild-type DAAO has some drawbacks whichrestrict use of the enzyme in practice. Rational design approachis widely applied to improve properties of the enzyme, such asthermal stability and catalytic properties.

Earlier in this laboratory DAAO from yeast Trigonopsis vari-abilis (TvDAAO) was cloned in E.coli cells. The recombinant enzymewas obtained in active and soluble form. Site directed mutage-nesis of Cys108 residue in TvDAAO resulted in mutant enzymeswith changed substrate specificity profile. Two mutant TvDAAOsshowed increased catalytic efficiency towards Cephalosporin C andone mutant had higher thermal stability compared to wt-TvDAAO.Two crystals of the mutant TvDAAO were obtained and the X-raystructure of the enzyme was determined at 2.8 and 1.8 A resolution.That provided us ability for rational design of TvDAAO properties.

Analysis of the TvDAAO structure showed that there are twoseparate enters to the active site of the enzyme for D-amino acidand oxygen, respectively. It was found that Cys298 residue isplaced in the smaller enter for oxygen and the sulfur atom is inSO3

− form. This finding correlates with data of Schraeder et al.(Appl.Microbiol.Biotechnol. 1996) about essential role of Cys298 forenzyme activity. Cys298residue was substituted for Ala and mutantTvDAAO Cys108Ala was used as template. Double substitution,Cys108Ala/Cys298Ala, resulted in crucial loss of enzyme ther-mal stability. Additional substitution was introduced to improvethermal stability of the double mutant. New TvDAAO with threemutations had the same Tm value as wild-type enzyme (54 ◦C). Thismutant and wild-type TvDAAO showed different kinetics of ther-mal inactivation. Activity loss of wild-type TvDAAO is describedwith sum of two exponential functions, while dependence of resid-ual activity of mutant TvDAAO fits to one exponential function.Production of new mutant TvDAAO with improved properties isin progress.

This work was supported by grant 08-04-01703 from Rus-sian Foundation for Basic Research and grant “U.M.N.I.K.”(N.V.Cherskova) from the Russian Foundation for Assistance toSmall Innovative Enterprises (FASIE).

doi:10.1016/j.jbiotec.2010.09.632