Apoptin gene therapy in head and neck cancer Schoop, R.A.L.
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(2) 3 Apoptin enhances radiation induced cell death in poorly responding head and neck squamous cell carcinoma cells Remilio A. L. Schoop Elizabeth M. E. Verdegaal Robert J. Baatenburg de Jong . . .
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(4) Abstract Treatment of head and neck cancers is still rather poor and worldwide new treatment options are sought. Sensitizing radioresistant tumors by combining irradiation with other therapeutics to induce apoptosis are widely $% & % ' ( $ %$% ) ) ) ' %$*++ % +% $,-/ % % ,01" % % ( 2 3 ) % % ) ) resulted in analyzed mitochondrial cytochrome c release and in cleavage of )453'% +,01" %% % did not. Moreover, in comparison to the irradiation only treatment the synchronized treatment of apoptin and irradiation resulted in increased cell %)% ,01"3 % + a colony survival assay. Our data reveal that apoptin treatment represents an effective way for enhancing radiotherapy of tumors responding poorly to radiotherapy.. 42.
(5) Introduction The worldwide incidence of head and neck squamous cell carcinoma
(6) ,# % 7!! !!! 1. Despite advances in % %74 $$+ ) % + 759 % $ 7-9 % 2. ; $ % % ( ) 3 radiotherapy and chemotherapy and combinations thereof. The prognosis of ) 43 ) 4 % ) ) improved during the last decades in part due to combinations of chemotherapy and radiotherapy5. In spite of these advances ) +
(7) ,) 3 part due to the resistance of cancer cells to radiotherapy and chemotherapy4. Lately, gene therapy has been successful in treatment of head and neck and other cancers via induction of apoptosis5-7. The apoptotic pathways are controlled by several pro-apoptotic and anti-apoptotic proteins, which might be defective or over-stimulated in cancer cells, respectively. Such imbalanced apoptosis machinery might cause resistance to radio-/ )3 $ )*) % ,$ of these gene therapeutic agents are even known to sensitize chemotherapy and radiotherapy8,9. Apoptin, a chicken anemia virus derived protein has shown to effectively kill tumor cells both in-vitro and in-vivo10. Cell death by apoptin )75%)%11 and normal cells are not affected by apoptin12. The level of impact in the apoptotic pathway seems to be different in comparison to radio- and chemotherapy/5.. 3') ur studies on the anti-tumor effect of a combination of radio-and apoptin gene therapy in radiosensitive and radioresistant squamous head and neck carcinoma cells. The combinatorial apoptin% *++ ( +% squamous head and neck carcinoma cells.. 43.
(8) Materials and Methods Cell culture. % $,-/% % ,01" head and neck squamous carcinoma cell lines known upon irradiation % * %14 are a kind gift from A.C. Begg, Netherlands Cancer Institute, Amsterdam, the Netherlands. The cell lines ' %1 K %*%PK% 1P# ))% ' /!9 + + 3 ) % ) Q+ Technologies, U ($3% # ' %5WoC and passed )X ' %+ $ )' ' ) ') Viruses and virus techniques. % $ $ ) ) ) Y%Q ) )# % Z4 % Y%[ Q\# $ % % ) $'%%% previously15 $ ') *%% 4% centrifugation. Titers of the viral stocks were determined by plaque assay on "// [ '2 %% % ++ /]!
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(10) ^]3/ 7_ ^]3 ! 3%79 #4`!j The cells were infected with the recombinant adenoviral vectors at a MOI of 5, which revealed under the used conditions a near complete transduction +*15. Irradiation. P ) ' ' %% /!4 % -4' ) '4+ 3' ) % $ Y%Q ) ) Y%[ Q\^ +75Wj ;% Q#'%%% '% ' %+ ] One day after the viral infection, cells were irradiated using a 6 MV linear. 44.
(11) accelerator. Doses were given in a single fraction of 2–6 Gy. After irradiation, the dishes were immediately returned to the incubator. Cytochrome c assay. ;%' + % 6. /! |/!4 %# ' % ' ' 4 % ,3 )% % + % !!!+ 7 ' )%% ++ 5!! 3/! )}) W ]~37!_37PY3 5 mM MgCl23 / % 3 / ) + %3 % /!! #3 + + 5! 3 % X% + $ ( % ' )% by centrifugation for 5 min at 2000 g $ ' $% from the resulting supernatant by centrifugation for 5 min at 14000 g. The resulting supernatant is the crude cytosolic fraction. All samples were frozen in liquid nitrogen and stored at -80° C until analysis by % % % *%4) % ) ,1,4YP#
(12) 3 ) c levels were determined. Equal amounts + ) were boiled in reducing sample buffer for 5 min, %% + % % % *%4/79) % ,1,4YY# gel, and electroblotted onto Immobilon-P membranes ) 3 %+ %3 Y3,Y# ' (%+ / ,4 ++ /! 4 3 ) W ]3 /7!
(13) 3 ! !79 '4 !# 79 + dried milk. Subsequently, the blots were then incubated during overnight ' %W ` / % //!!!# % ; ' $ 3''%% incubated for 1 h at room temperature with secondary antibody. Membranes were washed three more times and incubated for 5 min at room temperature ' , ) , U # $ ' $ X% by enhanced chemiluminescence according to the manufacturer’s protocol Y3U %3
(14) %#. 45.
(15) Caspase-3 analysis. ` /!5|%# ' ' -!4 ) % % follows: Cells were collected by gentle scraping, washed once with ice % ,3 % % 4 ++ ,3 /9 4/!!3 /!9 # ) U 1 3 %) 3
(16) 3,Y# Insoluble material was removed by centrifugation at 2,000 g for 15 min at 4°C. Protein concentrations were determined using the BCA protein ( 3 3 U (+ %3 Q3 ,Y# % + K ;+ + ) ) ) ' + %
(17) YP ]/ 9 [ 3 %3 Y3 ,Y#%+% 4U%3[%
(18) %# ; ' +3 ' (% + / ) 4 ++% ! /9'4 !%79 nonfat dry milk and incubated overnight at 4°C with monoclonal anti-active )45 % C92-605 % /7!!! 3 , 1 3 Y3,Y# ; % +% described for the cytochrome c assay. Clonogenic survival. The effectiveness of the combination of apoptin and ionizing radiation was assessed by clonogenic assays , '% 'Y%Q ) ) Y%[ Q\ (4+% '4+ % ' +% % ' 4 ) % ) % %++% of ionizing irradiation, and incubated for another ] 5Wj
(19) 3 ' )X% and counted. Known numbers were then replated in 10-cm dishes and returned to the incubator to allow macroscopic colony development for 12 days. The individual colonies ' * %%% ' ! 79 $ % /!9 + /! 7! ' scored and the percentages of surviving cells were calculated. The relative surviving fraction after each treatment was calculated based on the survival. 46.
(20) of the fraction of non-irradiated control treated cells, which was set arbitrarily /!!9 . Results Apoptin induces apoptosis in both radiosensitive and radioresistant HNSCC cells. ') )X% )
(21) ,3 we have analyzed its effect on irradiation of the radiosensitive SCC61 cells % + % ,01" ;3 ' $ X% $ + ) )4% % % '
(22) , ' ) )4% %%)%%
(23) , in comparison to radiosensitive SCC61 cells. ,-/ % ,01" ' % ' Y%Q ) ) % ) ) ' $ Y%[ Q\ % Z4 % ; % + + 3 ' * % % ) + ) ) % Z4 % ' X% % )* % 1
(24) Y + ' stained with DAPI, which stains intact DNA regularly but DNA of dead cells irregularly16. Apoptin-positive radiosensitive SCC61 cells contained for W-9%1
(25) Y ) ' % + W/9 + ) )4) $ % ,01" ; /# ^$4 ) + Z4 % % % $ + /]9 % /59 + ,-/%,01" ) 4 ) W9%`93 )$# + 3' % % $,-/ %% ,01" $$ ) )4 induced cell death. One might assume that the blockage preventing radiation% %%% ,01" $% apoptin.. 47.
(26) 9) ) . Figure 1. 90 80 70 60 50 40 5! 20 10 0. Apoptin. LacZ. Y) ) % % % $ ,-/ # % % 4 ,01" ) # $ ' +% ' Y%Q ) ) + )# %) ) '$ Y%[ Q\ )# % 4 ) ) Z4 % ; % + + 3 ' * % % X% % ) + ) )4 Z4 %4) $ that stained abnormally with DAPI is given as a relative measure for cell death induction. ' %)% )'% )3/!!' ) )4 Z4 %4) $' %. Combinatorial effect of apoptin and radiation on the survival rate of radioresistant SQD9 cells.
(27) 3'%%') )$ of tumor cells to radiation resulting in a decrease of clonogenic cell survival. ,-/ % ,01" ' ) % Y%Q ) ) $ Y%[ Q\% /%%%'!3 3] - + radiation. After another day, the cells were plated and incubated for 12 days to determine the colony formation as described in the Materials and Methods section. % 4) $+ ,01"%,-/) % ') ) Z4 %)%; 2. In comparison with the radiosensitive SCC61 cells, radiation induced % ) % ,01" ; 3 - radiation and Z4 % +,-/ % $$ + )) /!9 ; Y#3 ' + ,01" $$ + 5!9' $% ; # At the different radiation dosages, the radiosensitive SCC61 cells. 48.
(28) revealed a slightly enhancing effect of combined radiation and apoptin treatment in comparison to irradiation combined with the negative Z4 % ; Y# 3,01" '% additional ++ + ) ) % ' % ; 3 ,01" cells treated with apoptin and 6 Gy radiation revealed a colony survival rate +)) /!93',01"%'-% % the negative Z4 % '% +5!9 ; # ) )X% ,01" radiation or that combined radiation and apoptin treatment has an additive ++ %
(29) , ,01" Figure 2 A. 100. , $$ 9#. 10. 1. 0,1. B. 0. 2. 4 1 #. 6. 0. 2. 4 1 #. 6. 100. , $$ 9#. 10. 1. 0,1. Apoptin enhances irradiation-induced cell death in radioresistant HNSCC cells. Human radiosensitive SSC61 (panel A) and radioresistant SQD9 (panel B) cells were either irradiated alone (—) or irradiated and infected with AdMLP.apoptin encoding apoptin (- - -)
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(31) ! " survival rate was calculated by setting the non-irradiated control-treated colonies at 100%, #
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(37) ! $& separate experiments.. 49.
(38) Apoptin sensitizes apoptosis pathways in radiation-treated SQD9 cells. Besides the effect of combinatorial treatment of radiation and apoptin $$3 ' % ' ) ) sensitize the activation of apoptosis pathways in irradiated cells. Therefore, '$%')45$% + % %%%+) ) Z4 % combination with irradiation. Both radiosensitive SCC61 and radioresistant ,01" ' +% ' Y%Q ) ) Y%[ Q\ % subsequently irradiated with 2 Gy, as described in the Materials and Methods ; %3$ +)45% ) ) % Z4 % ' $ % ) ) separating equal amounts of cytosolic protein on PAA-SDS and by means of & )* %%%)45 cytochrome c, as described in the Materials and Methods section. U% $ ,-/ %' )45 $ $ X% )) + /W4(1 $ + )45 and cytochrome c release after irradiation combined with apoptin or with Z4 % ; 5# Figure 3 SCC61 apoptin & Q\ radiation. A. ,01" apoptin &. radiation radiation. Q\ radiation. Cytochrome c. B $%)45 Y) ) %'% $ +)45% + % ,01" %%,-/%,01"'infected with AdMLP. Y) ) '$ Y%[ Q\ Y %,-/%,01"X% & + % c )45$ %,-/%,01"'X%% +$%/W4(1$)45 . 3 % + % ,01" % ' Z4 % ) %% %$ +)45 ; 5Y# + + % ; 5# . 50.
(39) '$3 +,01"') )%% % $ +)45 ; 5Y#% + ; 5# '% +% ,01" + +* % ) ) )'3 )' $% ) %% +) ) %%$ ++ $%%% $$ ; # . Discussion Resistance of head and neck squamous cell carcinoma to conventional )3 % )3% 2 %%*) ) program17. The present in-vitro studies showed that the tumor-selective ) ) % ) ) ) %%$ ++ human squamous carcinoma cells treated by irradiation. By means of clonogenic survival assays, concurrent treatment of ,01"% '% %) )') $ an increase in apoptotic cell death in comparison to irradiation alone. The achieved cell death level reached almost comparable levels to those in radiosensitive SCC61 cells, which suggests that apoptin sensitizes the % ,01" % ) % *%% +% $) 4) ) + )45 % % % ,01"3 ' % ) % ') ) synthesis. These results suggest that in contrast to radiation, apoptin can $ ) ) ,01" + % % 2 $ +)45 Caspases18 play an essential role in programmed cell death, in which )45 +) ) '%)% % + ) ) % )45 $ 19. Besides. 51.
(40) this activation, apoptin-induced apoptosis results in cytochrome c release subsequent PARP-1 cleavage and Apaf-1 activation20. All these apoptotic ) $% ) ) * + % 4% ) ) ,01" '$3 + aspects remain unknown. Cancer cells typically evade apoptosis, partly due to an inhibitory mechanism on the mitochondrial cytochrome c release and subsequent $ +)45 % %% % % ,01"3'%%% $,-/ &%,01"%'% ',-/ cells, for they are both sensitive to apoptin-induced apoptosis? Strikingly, apoptin can induce selectively apoptosis in a rather broad range of various types of cancer cells21,22. + ) )4% % ) ) unclear. The fact that apoptin induces apoptosis in radiosensitive and %
(41) , 3 ) ) induces apoptosis in a different way than radiation does. Apoptin can induce ) ) + )) )75 3 5, whereas it is known % $$)7524. Recently, it has been shown that apoptin % )) ) Y|#' ( %)754%)% % % ) ) + + + 3'$ )75% are therefore resistant to many forms of anticancer agents25,26. In addition, various reports have shown that apoptin can induce apoptosis in cell lines ) 4) ) ) $$3 4 3 4 Q % Y16,27,28. These circumstances are known to inhibit anticancer treatments such as radiation and chemotherapy "35!. All these features reveal that tumorrelated blocks, within the apoptotic machinery, negatively affect conventional ) + $ + ) )4% % ) ) ; studies have to be carried out to unravel the precise mechanism of apoptininduced apoptosis and its role in enhancing radiation-induced cell death.. 52.
(42) The enhancement of radiotherapy with apoptin seems a meaningful option of further clinical investigation. Several studies have shown to increase survival and local control when patients are treated with the combination of chemotherapy and radiotherapy. The additive effect of ) * + + head and neck cancer5/ % ) % *++ + apoptin and radiation in radioresistant tumor cells, also other studies have ) % * ++ + + ) ) ' other therapeutic agents. Recently, Olijslagers et al. reported an additive ++ +) ) ) % ) %%) 3%)% +)75 5. In addition, Lian et al. analyzed the anti-tumor potential of simultaneous apoptin and interleukin-18 gene transfer in C57BL/6 mice bearing Lewis lung carcinoma5 . They reported that the growth of established tumors in mice immunized with plasmid pAPOPTIN encoding apoptin in conjunction with plasmid pIL-18 encoding (4/` ' * % )% ' ' + tumors in mice immunized with plasmid pIL-18 or plasmid pAPOPTIN alone. Liu et al. have observed that addition of the acid ceramidase inhibitor LCL204, in combination with apoptin, augmented tumor-cell killing under in vitro conditions28. This effect was also demonstrated in vivo in that apoptin % QQ !] 4 * % % ' 1/]7 + ^ ) % % % ) ) promising therapeutic agent for various types of cancer and that its function is improved when combined with relevant anti-cancer age.. Conclusions ^ % % % + * ) ) % % $ + %%(# % and apoptin treatment harbors potential for being used as a therapeutic )) $ % +
(43) ,). 53.
(44) References . . . . . . . . 54. / ( 13 ;3 ; 3 3 !! Y Cancer J Clin 2005; 55: 74-108. 2. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer !! 7 54]W 5 ; Y3_ &3 Y3,%(1 %%(
(45) P % !!/5]7/`"!4/"!! ] 3 \ 13 %
(46) ^$ ) ) ) Y%$P )% !!`-/7/!74/ - 7 P% 3 P% 3
(47) Y% $ )75 ) squamous cell cancer of the head and neck region. Curr Opin Mol Ther !!57-//4-/W - , ) UY3 % U3
(48) Apoptin induces ) ) % !!`W/5-`4 /5W5 W Q ',&3QY& Y) ) !!! /]`74]"7 ` (3,%331 %P3U %3; 33 , X4^ ++_3 %&. Sensitization of resistant lymphoma cells to irradiation-induced apoptosis by the death ligand TRAIL. Oncogene 2001; 20: 2190-2196. " Q&31 3,Q33 3Q,13 . Adenovirus type 5 E1A sensitizes hepatocellular carcinoma cells to gemcitabine. Cancer Res !!5-5- "4- 5- /! 13, 3&\33\ 3
(49) 30 ,. Inhibition of hepatocarcinoma by systemic delivery of Apoptin gene via the hepatic ) ) !!W/]--4W5 // \ 3Y3$%PY3
(50) $) Apoptin induces apoptosis in UV-C-irradiated cells from individuals with $ % 4) % U /""" 7" 5!/!4 5!/7 / ^ 3 1Y )* ) 4) ) + ) ) [)5# from chicken anaemia virus. Curr Drug Targets 2004; 5:179-190. /5 (3% ([3 3[(,3[3 %& Y) ) 4 modulating agents in combination with radiotherapy-current status and outlook. Int J Radiat Oncol Biol Phys 2004; 58: 542-554. /] ,;3 P 3Y%4&Y 3( 3Y 1++ repair of radiation-induced DNA damage in cells of human squamous cell carcinoma and the effect of caffeine and cysteamine on induction and repair of DNA double-strand breaks. U%U/""]/]!/754/-!.
(51) . . . . . . . . /7 $%P3Y3,);3_ ))3$%[%3
(52) 3 U Gene therapy with apoptin induces regression of +% ) !! "754-/ /- , ) UY3 _ _3 % U3
(53) 4 Q )754 ) )4% %) ) %%(2 !!]/!"5`4] /W ; %,31_ ) ) )') Cancer Drug Targets 2004; 4: 569-576. /` ;3, X4^ ++_ Y) ) 4%)%% Cell Death Differ 2005; 12 Suppl 1: 942-961. /" 14$ ^ YY3 $ % P Y3
(54) The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol 2000; 74: 7072-7078. 20. Burek M, Maddika S, Burek CJ, Daniel PT, Schulze-Osthoff K, Los M. Apoptin-induced cell death is modulated by Bcl-2 family members and is Y)+4/%)% ^ !!- 7 /54 /
(55) Y) ) 4)* ( ) + anti-tumor therapy.
(56) 44%# !!77/]"4-! (% +3[YP3% Y3\U3[3[ ()3 \ 3
(57) Apoptin: therapeutic potential of an early sensor + + Y U$ !!` ]` /]54/-" 5 ^,3\ 3(% +3
(58) Y%%$ ++ +) )% ) ) % ) % !!W/!!/ W4/5/ ] ,_Y3P1&, )753UY/% Y%$ P )% !!W-!`W!4`- !W 4W!W` 7 1&3 % 3U Y) ) ) 2 % + )4)* X 3 ) ) 3 % + )4) ) | Q % [ !!-`!W7574W7]7 26. Russo A, Terrasi M, Agnese V, Santini D, Bazan V. Apoptosis: a relevant tool for anticancer therapy. Y^ !!-/W3, ))W$//74$/ 5 W 14$ ^ YYY3 [ % P Y3
(59) Bcl-2 ) )4% % ) ) Y%$ P ) % /""" ]7W ]74 249. ` Q 3P ,3P4\'Y3 1 3'(Y % + % $ ) ) )K ) !!-/]-5W4-]-. 55.
(60) . . 56. 29. Gimenez-Bonafe P, Tortosa A, Perez-Tomas R. Overcoming drug resistance by enhancing apoptosis of tumor cells. Curr Cancer Drug Targets 2009; 9: 5 !45]! 5! 3
(61) 3 % ' $$ , 2008; 99: 1709-1714. 5/ ,3 \ 3 , 3 , 13 \ ' )% '+ ) 4% 4%%)' and its implications for the chemoprevention of head and neck cancer. Mol Cancer Ther 2005; 4: 1448-1455. 5 Q 3
(62) 3Q33\3\3, Q3Q3\ 3Q. Induction of an effective anti-tumor immune response and tumor regression by combined administration of IL-18 and Apoptin. Cancer Immunol Immunother 2007; 56:181-192..
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