SPHEROID 5FU - St. Marianna University School of...

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� � ����������Vol. 32, pp. 283�290, 2004

������ SPHEROID ���� 5FU ���������5FU � ��������������

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� �� � TGP �Thermoreversible gelation polymer� ����� !�" 5#$%&'()*

� 3+,��-. spheroid �/0-� 5FU 12345 5FU 67#��8'9:;<2=>? OPRT �Orotate phosphoribosyl transferase�, TP �Thymidine phosphorylase�, UK �Uridinekinase� @ABA%;<2�CD-� 1� 7#��8'9:;<2% 5FU %E�F!GH?IJ� 2� OPRT KFL6 5FU E�F6%MN� 3� 5FU ��8'OP%QR/�!S".TU-V� @%WX�1� OPRT ;<Y �OPRTI� � OPRT KF�Z[?&'()*=� 5FU %\X�]^_`V� OPRT KF%a" Paca-2 =�\XbcdAaefV� TP ;<Y �TPI� � OPRT%KFL%g" PC-9 �hi()*&�� MEC �jk)*&�� Lovo �li(&�!3m?5FU%\X�Zn!op-V� UK;<Y �UKI��q.%&'()*!3". 5FU %\X!IJ-aefV�

2� 7#()*&% OPRT 9:KFL%g]6 5FU %E�F6%r!�QMMNbstdAaefV�

3� OPRT KF�Z[?&'()*% 5FU ��8'uv�� w 1 OPbx=>y� w 2�w 3OP�z=>?� w 2OP�{|�/�-."a"6}~_AV� OPRT KFL%gL% PC-9, MEC, Lovo %w 3OP�;<[?6� viability b]^[?�6ed� w3OP� FU ����%���b��."?6�HdAV�

�������F������� �+,��� �(2E�F��

5-fluorouracil �5FU� ��(2%\X��!���9:=>? thymidylate synthase �TS�, ��8'9:=>? orotate phosphoribosyl transferase

�OPRT�, thymidine phosphorylase �TP�� uridinekinase �UK�, @-.uv��9:=>? dihydro-

pyrimidine dehydrogenase �DPD� a�� # %uvM�9:bee�f.3y� @%KF 9:¡!4y�¢£\Xb¤¥_A?� z¦� �Ad%9:§�¨©-.3y� QRMN�ª-"6_A."V1�� �A«=% in vitro =%TU=�&'()*��¬��-� @A!�­".9:KF%Z®� QMMN�¯°.�V2�3�� -e-�¬��=%)*±²��³´6qµ¶a?·+,±²=>y� �V-.�¬��=¸dAVWXb@%««�³´=%��������� ¹�; º»¹�

283

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������������ ������������������� 4� �TGP; Thermorev-

ersible gelation polymer� � ����������� �!�"#$�%� ����&'%��(�� ����&)%��(��*���+��!�"#$�%��5��8�� ,-���./��0�1� 23 4�����56�� ���1�7�8��� � spheroid ��3 9:�� ��; �9�10�� ����%�6<3 = spheroid � >�?��@A�����B �CD%��(��E���1*��%�F�%3 = spheroid �GH �,1�%I�� JK�3 ��LMNOP=>�Q��RS�ITU��-<��1VWX-�� �RS%� ,�Y�* TGP ���� �6�Z[\#23 � spheroid �]!6� 1� Z[^M_#>�"`a� 5FU �# �3 �bc��$W�de� 2� OPRT Q�%13 � 5FU bc�1�fg� hYi 3� 3j� 5FU^M_#k&�'l]��j0�m(6<�

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�� ��1� n�3 \DLD-1 �)*+23 \� � ),-.op\q

rs,/0�tRSu� 12345v�wxy�Yz{6|-<� Lovo �)*+23 \�� PC-9 �7+23 \� � }~��RSuYz�$|-�����8�6<���n�6<� MEC �9�3 2\� � �:X11���;6<� CA19�9 ����9�3 2<=�\#23 %��� MIAPa-ca-2 �>�3 23 \� � ?@)��ARSu��B3�5vYz�$�cD<� )� 5[��3 \� 10� fetal calf serum �FCS, Invitrogen Co.Ltd. Scotland, UK� �� RPMI 1640, penicillin

�100 unit�ml��streptomycin �100 mg�ml� �Life Tech-nologies, N.Y. USA�, L-glutamine 2 mM � l �LifeTechnologies� ��W�����0 37�C� 5�CO2 )���'%�6<�2� n�-a�2a16� 5FU ��4C>op\qrs��n�6<�OPRT "`a%�� Monopotassium 1,2,3,4, -tetrahydro- 2,4 -dioxo- 1,3,5 -triazine- 6- carboxylate

�OPRTI�, TP"`a%�� 5-chloro-6-�1-�2-imino-pyrrolidin- 1-yl� methyl-2,4 �1 H, 3 H� -pyrimidi-nedione hydrochloride �TPI�),-.op\qrs�?��Yz�$�cDn�6<� UK"`a%�� 2.6 dinycroxypyridine hydrochloride �UKI� �f?#�\qrs� ?���D6n�6<��� �1� TGP �];�TGP��X�E�12�13��YK�];6<� ,�TGP FG�)�����W 4�C '%�H��6� ��I� 10� �wt�wt� �J�6<�2� Z[\#23 � spheroid ];�24well�~�� flat bottom multiwell tissue cul-ture plate �FALCON, Becton Dickinson Co.Ltd.Franklin, NJ, USA��� 4�C�B TGP �������(���Z well � 300 mlAjKD6<� Z\#23 � 1.0�105��ml�3 L��*�Y��4�C�B��%J�6� Z well � 100 mlAj�K6<� ���� 4�C'% 30� ¡¢��H���(��6� 3 £M��6<� ,� 24well �~��� 37�C¤¥��~��)�NO6� TGP ����#6<� ���¦P6<(�%���Z well� 600 ml Q�6� $§ 1000 ml �6� 37�C�5�CO2 )���'% 5R �6<� Viability SP�¨ �E�%SP6<�OPRT >�Q�%SP���©� spheroid �TU*<U� �-ª-�\#23 ��B TGP ���3 L�� 2.0�106�ml �*�Y�� 5 ml ]!6� 75 cm2�«�KD6<� 37�C¤¥��~��)�OI TGP ���#6<�� �� 15 ml�W� 37�C� 5�CO2 )���'% 1¬ �6<�spheroid �GH� 75 cm2 �«�V)�OI

�B6< PBS ��WE���6<� £M�� 2000rpm �83�10G�%­®�W6<�� spheroid�GH6� OPRT>�Q�SP% 80�C%X¯YZ6<�3� OPRT >�Q�%SPOPRT Q��SP� Shirasaka X14��E��JK� �6�14C� 5FU ��0< radioenzymatic assay�Yz°0� [\O�±�O²³´��Yz�;���W6�P]6<� µM���¤¶·¸�¹64�C� 60 �­®�W�105,000�g��� �6�14C� 5FU,PRPP �pyrimidine phosphoribosyltransferase� ��W� 37�C % 10 �� 30 �+|@� ^M_#�

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���� ������ 4�C� 5 �� �10,000rpm� �� � � HPLC �������� 5FU �FdUMP� peak���������� of total ra-dio activity ����� ����� !"#$��"#%�&��� 5FU �'(�� !)*+,������ )*+,- p mol�min�mg protein .�/�� ���0 5.0 pmol 12�% ND �not de-tected���3��� $�4- Bio-Rad� proteinassay kit �Bio-Rad Laboratories, NY, USA���5$����678 9:;<= �fraction V� ������4� 5FU >?,@ABCDE� Fig. 1 �/F��1� 5FU G�HIJK=LM)*NOP�QR

� �Fig.1�IJST ��� 2� . 5U�����VW�X

�I well ���Y 600 ml�Z[\]��� ^5.��Y 500 ml�QR�� �!�_`4 1 mg�ml�a��b�cd�� 5FU �� "e�fg- 50 ml�well � PBS � 50 ml�well Rh� IJK=LM)*NOP���fg- 5FU 50 ml�well �i4�IJK=LM)*NOP.j� OPRTI, TPI, UKI�k�l� 50 ml�well m^QR�gn 1000 ml �� 3U�G�H 5U�op�q���2� Viability ���rSP"es�-K=LM)*NOP�����3U�s�- 5U�op���IT � viability �WST-815, 16� ��6����� t�,� Formazann�u�F� WST-8 �2-�2-methoxyl-4-nitrophenyl�-3-�4-nitrophenyl�-5-�2,4-disulfophenyl�-2H-tetra-zolium, monosodium salt�8 �vwxyz{M|}~�� ����I well� 50 mlG�H 10����L�XK��� 50 mlRh 37�C. 6%����q���

����VW�X���n Labsystems MultiscanMS ��U���vwxy� ��56 450 nm ����� 620 nm� .Z�4������viability -��w������

T�C�����5FU ����"#��$��&���OD �optical density��������=�� OD����=X��:������"#��$��&��� OD�������=�� OD� 1005� �n�����-�6����� ¡.�/��� ¢£�¤�¥�¦� �One-factor ANOVA� ��56�¥�¦�� �§¡¨��- Fisher[s Protected LeastSignificant Di#erence�Fisher[s PLSD� ��5� p�0.05��§¡j���©��� OPRT +,�� vi-ability��ª«-� Peason[s correlation coe$cient�¨���ª«¬­���� �n|®�§,-Fisher[s r to z� !¯°����� p�0.05��§¡j���©����� ��1� IJvMST � spheroid �G±� OPRT

)*+,�IJvMST � 1²� TGP .���6u�����³� 100 mm´�� spheroid µ� OPRT )*+,�� Fig. 2 �/F� DLD-1: 22.4�1.0 �n�6�, Lovo: 74.2�4.4 �n�8�, MEC: 369.5�16.9 �n�3�, PC-9: 609.4�58.4 �n�3� �pmol�min�mg protein�.j�� MEC, PC-9 0¶�vMST v��·6�§�¸�.j�� �p�0.0001, p�0.0001�� Paca-2�)*+,-¹�!�a ���2� IJvMST spheroid � 5FU >?,�^56

IJvMST v� spheroid � 1 mg�ml �4� 5FU "eG�H 5FU �IK=LM)*NOP

Fig. 1. Procedure of chemosensitivity test.

5FU K=LM)*NOP�>?,º�»¼ 285

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����� 3��� 5���� �� viability�T�C���� ���� Table 1, Fig. 3 ����5FU ��� 3����� 5��������� �������� OPRT !"#$% viabil-ity %���&'�()*+,-./01��3� ������� spheroid ��23��456� !789� 5FU :�;�<= �Table 1,Fig. 3�

�1� OPRTI � 5FU :���>�<=5FU 1 mg�ml% OPRTI 1 mg�ml%����?��3��@A���3 PC-9 � viability * 5FU ���B��&'�CD�� �p�0.05� ��E���F������ spheroid � viability *&'�G�*+,-./01�� �0�� 5��@A���3% Lovo, DLD-1, MEC, PC-9 � viability * 5FU���BH��&'�CDI+,-.� �p�0.0021, p�0.012, p�0.0097, P�0.0072�� Paca-2J* 5FU ��%K>LM� viability JN1��OPRT !"#�&�3��J*� OPRTI �O

D�1� viability IP$����Q%0-�OPRTI * 5FU �R��:��78��� OPRT"#I+,-./� Paca-2 J*� 789* 5FU�:��<=��> /01���2� TPI � 5FU :���>�<=5FU 1 mg�ml % TPI 1 mg�ml %����S� 3����J* PC-9 � viability �TI 5FU ���viability �BH��&'�U$���� �p�0.0033�� 5����J* PC-9, MEC, Lovo � vi-abilityI 5FU���B��&'�U$���� �p�0.0091, p�0.0021, p�0.006�� OPRT "#�P�

��J* TPI * 5FU �R��:��CV����3� UKI � 5FU ��>�<=5FU 1 mg�ml % UKI 1 mg�ml ���J*� 3��� 5��@A����W�������� viabil-ity * 5FU ��� viability %BH��&'�G�*+,-./01��

� �

5FU17�*X�Y��E�3�Z[\����]W^� .��3_9JN318�� `�abc:��d�ef*� fluorouracil I456� .� 5-fluoro-uracil 5[-triphosphate �FUTP� %/S RNA �gShi.� RNA �ejk81922�%� 5 -fluoro- 2[-deoxyuridine 5[-monophosphate �FdUMP� Ilmno6pq !JN3 TS %rstu6%��Jternary complex �vq� DNA pq�78�3Q%Iwx-.323�� 5FU �456�yz�* 3 {�yzIN3% .��3� i|� } 1 * OPRT�3 5FU 0- FUMP ;�yz�~�� } 1 yz�JNS� } 2* uridine phosphorylase % UK

���3 5FU0- 5 - fluorouridine �FUrd�;� -� FUMP ;�yz�~�� } 2yz�� `��}3* thymidine phosphorylase % thymidine kinase�TK� ���3 5FU 0- 5 -fluorodeoxyuridine

�FUdR� �y�� -� FdUMP ;�yz�~��} 3yz�JN3� OPRT *����4mn5�a�9 �5FU� �"#����l��G��3 !J�DNA � RNA 78�)�3}������/ !%��-.��3� ��-3�*� OPRT �35FU�456�I 5FUabc:��������{JNS� i�`�yzI�������235FU 456����yzJN3%�����3�TP *�4mn5���������)��3 !JNS� lmn5%lm5���G��A��3�5FU *Q.-� !�S456�� 2� "#� .�abc:��¡¢�3� Q.-� 3{� 5FU 456�yz*� �£��456��¤¥�0� f¦IN3�0� N.§¨.I��yz/�0� `��©��(©d����3�0/¨�{��ª«�3�,� ¬­-*�yz�)�3 !�789��������� spheroid� 5FU�R��:��®¯���OPRT "#�/� Paca-2 �?�*� 5FU %

OPRTI ���°���W� viability * 5FU ��

Fig. 2. OPRT �orotate phosphoribosyl transferase� enzy-me activity in each cancer cell line. : p�0.05

±² ³ ´µ ¶286

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������������ ����������� OPRT ���������� !"#$%&��'�� Paca-2 $� 5FU �( 2� ( 3)���*�'�+� %&���,�)�$-./�0��&�12��&� 3'3� Paca-2$( 2� ( 3)�45�6� UKI� TPI $7839 viability ��� �������':; �Table 1���'���)��<=3'>?@����3���ABC&���D�0�;� EF� UKI, TPI $( 2� (3)�4GH�78C&���IJ� ����4K�C&L�$%&�OPRT ��4C&MN�O�� 1 mg�ml �

OPRTI � 5FU 4P=��C&�� 5QRS�$OPRT ��T�UV�WX�Y viability ������Z3[\]^���_`3;� ����'�( 2ab( 3)��<=�( 1)����c����D�0�;� d:� ( 1)��ef�3��gh3�&iY 5FU jk�lm�+n)�$%&���K�0�;� OPRT���%&MN�( 2)�4 UKI$7839 viability ��� �������':;��IJ� ( 2)��-./�<=�op�c�'� %&��<=3������D�0�;� ( 3)�4 TPI $78C&� OPRT��TUT� Lovo, MEC, PC9 � viability ���

���Z3��V#4q3;� ( 3)��rs3;t�Y FUdR4) FdUMP �uv0�&wx��y TPI �I: viability �_`C&���Lz0�;�� {m�|�:�;� TP ��T�TK ��TIJ9UT�}MN�$�� ( 3)��FUdR '� 5FU uv�~wx$%&���0��&2�24�� d:� ���� OPRT ��T�U�MN�$� TP ��T�U����D�0�� 5FUuv���~wx���:�&�12��;�������}MN� spheroid 4=�;{m$�� OPRT ��T� 5FU �����R��WWX������':;� OPRT ��T�UY'> TP��T�U�MN�� ( 3)�� 5FU uv���<=3� FdUMP �I& DNA �8���Y�:�&;����3�&� EF� OPRT ���U���}MN� TP ��4��3� ����4K�C&L�$%&�

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�4�2&�%;J,����t��4�:;��-�.����]����� �¡¢�£¤�&¥�4¦3§C� ¨©-./���78ª4«IYt¬­#0J� §; OPRT ���t®�;¯�;�°±²�³´µ�¶Y·¸3_¹§C�

Table 1. The E#ects of each enzyme inhibitors on the viability �T�C�� in each cancercell line. Upper column shows T�C��� on the day 3. Lower columnshows T�C��� on the day 5. All of T�Cs show mean�SEM. �: p0.05,��: p0.01 vs PC-9 cells. �: p0.05, ��: p0.01 vs day 3.

5FU -./���78ª����º�»¼ 287

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1� !"#� $%&'� ()*� $%+,� -.��/01 TP 2 DPD 34� �25�672003; 30: 495�500.

2� 8%9:;� <=>� 5-Fluorouracil �?@8AB� CDE�FDGHEAB� 1991; 15:112�118.

3� IJKL� M=NO� =PNO� QR�ST�/01 5-FUDEU5V!�WX� �25�67� 1996; 23: 721�731.

4� Heskins M, Guillet JE. Solution properties ofpoly �N-isopropylacrylamide�. J Macromol.Sci. 1968; A2: 1441�1445.

5� Y%Z� [\]� �^_� `ab� cdeEf\ghi�jkDlm �GMW� nop��qrsAtu2��ivjwx� ��yz{|}~� �����/01�R������� ���������� �� 9 �h������

Fig. 3�1. The e#ects of each enzyme inhibitor on the T�C�� in each cancer cell line onday 3. Error bars show SEM. The concentration of 5 FU and each enzyme

inhibitor is 1 mg�ml. : p�0.05

Fig. 3�2. The e#ects of each enzyme inhibitor on the T�C�� in each cancer cell line onday 5. Error bars show SEM. The concentration of 5 FU and each enzyme

inhibitor is 1 mg�ml. : p�0.05

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7� ���� ���� �� �� � ������������������ !!"#$%��&� '()*+,-./ �01234567897�:;<� =>?@ABCDE� FG 8H�?@G"IJ�� 1997; 37�42.

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11� ��t�� uvwx� y�z{� |}~� ����� ��w� b��W� �����: CA19�9�N��8�����d �MEC� ��������� HUMAN CELL, 1990; 3: 346�351

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5FU �� X¡¢£¤���¥�%�¦§ 289

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Abstract

The E#ect of 5FU Phosphorylation Inhibitors on the

5FU Sensitivity to Several Cancer Cell Lines

Yasushi Konnno and Satoshi Tsukikawa

Five types of established cancer cell lines were three-dimensionally cultured using thermoreversible

gelation polymer �TGP� as culture medium. Single agent 5FU or 5FU with kinase inhibitors of orotatephosphoribosyl transferase, thymidine phosphorylase, and uridine kinases were added to the medium to

investigate as follows; �1� the e#ect of each kinase inhibitor on the sensitivity of 5FU, �2� the correlationbetween OPRT activity and 5FU sensitivity and �3� the interaction in the three 5FU-kinase pathways. Weconcluded as follows;

�1� OPRTI had an inhibitory e#ect regardless of the OPRT enzyme activity level in each type of cancercell line and o#set the e#ect of 5FU. TPI inhibited TP of PC-9, MEC and Lovo that had high OPRT

activities and enhanced the e#ect of 5FU. UKI did not inhibit the cell viability of any cancer cell line.

�2� There was no correlation between the OPRT activity level of each cancer cell line and the 5FUsensitivity.

�3� The OPRT pathway is considered as the main route of the phosphorylation of 5FU, while the TP andUK pathway are altenative routes. Particullary, UK pathway is presumed to be inactive under normal

conditions.

General Surgery of St. Marianna University School of Medicine

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5FU ���������� ��� 291

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