,

t Life ScienceJournal, 3 ( 4 ), 2006, Ding, et al , SonosensitizationMechanism of A TX-70

~~,"~

Sonosensitization Mechanism of ATX-70

in Sonodynamic Therapy"

...

Chunfeng Dingl, Junhong Xu2

1. Henan Key Laboratory of Laser and Optic-electric Information Technology,

Zhengzhou University, Zhengzhou, Henan 450052, China

2. Faculty of Dynamical Engineering, North China University of Water Conservancy

and Electric Power, Zhengzhou, Henan 450002, China

,.,

Abstract: Sonodynamic therapy (SDT) is an effective method to cure tumors, but the mechanism is not clear up tonow. In this work, the mechanism of SDT was analyzed by studying the reactions of gallium-porphyrin analogue(ATX-70). Results showed that the ATX-70 might play two roles in SDT. One was that the high temperature

produced in the bubbles at collapse promoted ATX-70 into excitated states, and reacted with the dissolved oxygen inliquid and produced oxygen free radicals, which was thought as an effective killer for cancer cells. The other wasthat ATX-70 played a role as surfactant in the process of cavitations and eased the cavitations, leading to producemore high-energy hydroxide radicals. [Life SCienceJournal. 2006 ;3( 4) :85 - 89] (ISSN: 1097- 8135).

"L,

tl

,"

\

Keywords:sonodynamic therapy; ultrasound; ATX-70; cavitations

Abbreviations: ATX-70: gallium-porphyrin analogue; CL: chemiluminescence; DMSO: dimethylsulfoxide; IC-CD: intensified charge coupled device; FCLA: luuoresceinyl cypridina luminescent analog; PDT: photodynamictherapy; SDT: sonodynamic therapy; SL: sonoluminesenece

l

,i~

1 Introduction

~

Sonodynamic therapy (SDT) is a new methodto cure tumors[ 1~13]. This method, compared to

the photodynamic therapy (PDT), has two virtuesfor clinical application. One is that ultrasound has adeeper penetrability, and the other is that itneedn't avoid light in the whole process of treat-ment. The gallium-porphyrin analogue (A TX-70)

is a widely used sonosensitive in SDT[3J . It can se-lectively gather in the tumors and enhance the ef-

fect of therapy. However, the mechanism was un-known[4J.

There are different opinions on the mechanismof SDT. Umemura firstly thought the sonolumines-cence (SL) excitated the ATX-70 to produce 10zwhich can kill the tumor cells, the same mechanism

as PDT[5J. The study of Kessel et al pointed thatthe sonosensitive function of ATX-70 was relative

to the cavitation[6J. Miyoshi[3J studied the mecha-nism thoroughly and found that the oxygen contentin the gas bubble was important to the cavitation.20% Oz was necessary for sonosensitive process,and the ATX-70 maybe acted as surface activator tostrengthen the cavitations. He further indicatedthat the sonosensitive reaction did not cause from

the SL excitated by the sonosensitiver, but has its

,."-.

l

\

~\

!t.I

-

~~~,\~

~

\.{

own mechanism. There are always different view-points on whether the ~ takes part in the sonosen-

sitive process. The research of Sakusabe et al[?Jsuggested that the sonosensitiver can kill tumorcells by enhancing the yield of ~ and other active

oxygen in the process of cavitation. Yumita et alalso thought that the active oxygen produced in the

sonosensitive process was crucial in SDT[5J. How-

ever, in sonosensitive experiment, Miyoshi[8J et aldid not find ~ by EPR. In addition, it's not surewhether the ATX-70 can be used repeatedly inSDT. At present, there are no detailed reportsabout these problems.

In this paper, mechanism of the SDT wasstudied by optical method. In the experiments, theselective probe of active oxygen named fluoresceinylcypridina luminescent analog (FCLA) and un-selec-tive chemiluminescence (CL) probe named luminalwere to detect whether the active oxygen producedin the sonosensitive process timely and directly.

2 Materials and Methods

2. 1 Reagent preparationFCLA (Sigma, USA) was diluted to

50 fLmoliL by distilled water. It has been knownthat FCLA only reacts with 10z and.o2 [14,15J. Lu-minol (Sigma, USA) which can react with many

~

. 85 .

3

rt

J)1

1

J

J.

Life ScienceJournal, 3 (4), 2006, Ding, et al, Sonosensiti:mtionMechanism of ATX-70

sorts of free radicals and emit photons[16], was di-luted to 50 /lmol/L by distilled water. ATX-70(Toyohakka Kogyo, Japan), which was thought asthe best sonosensitizer[3], was also diluted to 50/lmol/L by distilled water. SOD (Sigma, USA) se-lectively consuming 02 , was diluted to 10 /lmol/Lby distilledwater. NaN3, a sort of medicament toreacting with l~ selectively, was prepared with 10mmol/L[17], and DMSO, reacting with 'OH selec-tively, was prepared with 10 mmol/L.

When the experiment began, injected somedistilled water in the glass firstly, and then addedthe needed reagents, made the whole volume 2 mland the concentration of FCLA, luminol, ATX-70, DMSO, SOD and NaN3 was 1 /lmol/L, 1/lmol;L, 2 /lmol/L, 2 mmol/L, 1 /lmol/L and 2mmol/L, respectively.2. 2 Apparatus and methods

The experiment setup was shown in Figure 1.The SL or CL was detected by: intensified chargecoupled device (ICCD) image system(Princeton In-struments, ICCD-576-S/1, - 40 "C). The ST-130controller(Princeton Instruments, USA) controlledthe ICCD and put the optical signal into computer.The luminescence intensity could be caught by thesoftware of WINVIEW. The ultrasound field madethe reagents mixed equably and quickly.

The signal function (AFG320 , SONY,Japan) brought sine signal with 500 kHz and fedthe signal to power amplifier (ENI CO. Ltd,21O0L, 50-dB). The 50 W signal from the amplifi-er was used to driver the transducer (diameter: 5cm, Mingzhu, Guangzhou, China) to emit ultra-sound. To avoid the influence of idle photons, theblack ink was used as the transmit media of ultra-sound, and the temperature was kept 25:t 1 "C.

The absorb spectrum of JITX-70 was mllected byabsorb spectrum system with the split of 10 nm, whichusually was used to highly sensitivedetection.

Da!kroom

Sample5T-130 controleJ:

BJadt idt

..Figure 1. The setup for CL detection

Results and Discussion

Figure 2 is the detected curve of CL intensityin different solutions, with the power of ultrasound50 W, the frequency 500 kHz, and the total time50 seconds. In the first five seconds, the back-ground intensity of ICCD is about 50 cps, andwhen the FCLA was added (the second five sec-onds), the intensity increased to about 450 cps. Itcan be taken as the experiment background. Afterthat, when the ultrasound began to act on the solu-tion, the CL intensity increased to about15,000 cps. When ATX-70 was added in, the in-tensity increased to about 45,000 cps immediately.However, with the SOD was added in, the intensi-ty decreased to about 21,000 cps, and it would de-crease ulteriorly to about 2,600 cps when NaN3 wasadded in. The experiment was repeated thr~etimes, and the standard deviation at each timepoint was lower than 9% .

)JIJf

l

J)

J

)..t

.I

1j

NX.1Q SOD

.. ~ ~

~+

4.

J1

I~ N-~ Ultt- 1 ~I.. ~ 11W~::;

3J~- - - L<.> ."--

,. Fr.

~~~

I;;;,~j.J

ii~I

,. 20 30Time(3)

40 50

Figure 2. The CL intensity of saturated air liquid in 50 sec-onds. The data was means:tS.D. (n =3)

J.

j

As we know, FCLA can react with active oxy-gen selectively and emit photons. When the ultra-sound appeared, added the ATX-70 to the FCLAsolution, and the CL intensity increased obviously.It was believed that it was ATX-70 that acceleratesthe production of active oxygen radicals. When theSOD was added in the solution, the CL decreasedbut not disappeared, which indicated that not only02 but also other active oxygen radicals were pro-duced in the process, for the SOD only react with02 and decrease the CL. The CL intensity continu-ously decreased when the NaN3 was added in,which further proved that the l~ radicals wereproduced in the ATX-70 solution. The final inten-sity was 2, 600 cps not 450 cps, which indicatedthat the ATX-70 not only participated in the pro-duce of active oxygen but also promoted the produc-

t~J

/.fjJ

f

(

f

'"

. 86 .

,,J

$

J1)

~

Life ScienceJournal, 3 (4 ), 2006, Ding, et al , Sanosensitimtion Mechanism of A TX-70

tion of sonoluminescence directly.Figure 3 showed the dependence of CL inten-

sity on the gas component in liquid. The leftshowed the CL intensity under saturated air condi-tion and the right under saturated Nz condition.The (D) and (~) represent the CL intensity ofFCLA and FCLA + ATX-70 solutions, respective-ly. As shown in Figure 3, the CL intensity ofFCLA + ATX-70 solution under Nz saturated condi-tion was only about 1, 000 cps, much lower thanthat in the air saturated solution, which wasreached to about 50,000 cps. It is because that inthe air saturated solution, ATX-70 reacted with thedissolved (h, and produced l(h, and (h, - . However,in the Nz saturated solution, the ATX-70 can't re-act with (h" and no active oxygen could be pro-duced. It indicated that, in the cavitations, ATX-70 can't produce the active oxygen free radicalssolely, without the dissolved oxygen. Thereby thecontent of oxygen would affect the CL intensity.

60

~ 50

~§ 40-3~ 30

'S 20

~d 10

0 FCLA

mI J!IX-70+FCLA

0

.tile NiIrogtnGases

Figure 3. The CL intensities of 1 f.LMFCLA(D) and FCLA+ ATX-70 (~) solutions which were detected in the airsaturated (left) and nitrogen saturated conditions (right) re-spectively. The data was means:t S. D. (n =3)

-

Figure 4 showed the absorb spectrums ofATX-70. Curve A represents the spectrum whichwas detected before the ultrasound and curve B rep-resents the spectrum 20 seconds after the ultra-

. sound. Before and after the ultrasound, the twoabsorb spectrums of ATX-70 were similar. Thepeak values of the both spectrums are about 400nm, and the subordinate values are about 600 nm.The result showed that, in spite of the ATX-70 ac-celerated the active oxygen produced in cavitations,however, its molecular structure was not de-stroyed. In Figure 4, the intensity of curve B de-creased more slightly than curve A at 400 nm. Thereason is that ultrasound makes the ATX-70 redis-tribute in the solution and leads the scan light ofspectrum system not to irradiate them completely.

~

\

From the above experiments, it can be con-cluded that, with the dissolved oxygen, ATX-70promotes the production of active oxygen radicals inthe process of cavitations. To describe how theATX-70 acts in cavitations, luminal (mainly reactswith' OH) was added in the following experi-ments. The sequence of the reagents was shown inFigure 5.

No AUtt..oUhd ~

B

fj.;!

\~--.00 .00 ,..'.0'00

W ""."O'h (nm)

Figure 4. The absorb spectrums of ATX-70 before and afterultrasound act. curve A: spectrum of ATX-70 before ultra-sound act; curve B: spectrum of ATX-70 after ultrasound act20 s

00 DMSO

so

1[ '0c!!Ico~.3 302!>"inIi~ 00....<..>

{JJauo,,",

.0 ~ Water

+0 .

0 .0 00 30 10

Tim .Co)

so .0

Figure 5. The change of CL intensity in luminal solution forprove the production in cavitation. The data was means :tS. D. ( n =3)

-When the luminal was added in the water,with the ultrasound, the CL intensity increased ob-viously. However, when DMSO was added in, theCL intensity decreased quickly, just above thebackground. Then, adding the SOD and NaN3 inorder, the CL intensity did not almost change. Itindicated that it was' OH that was mainly pro-duced in the process of cavitations, but the yields ofl(h, and (h, - were very low. When adding ATX-70into the luminal solution, as shown in Figure 6(the experiment was repeated three times), the CL

L

. 87 .

sonosensitizer in SDT.

I

1~

1

tfI

f}J

.\IJI

f1

Life ScienceJournal, 3 (4), 2006, Ding, et aI, Sonosensitimtion Mechanism of ATX-70

intensity decreased from about 51,000 cps to about42,000 cps. Adding FCLA into the mixed solu-tion, however, the CL intensity increased again. Itindicated that the ATX-70 can react with the 'OHradicals and decrease the concentration of the' OHin the cavitation, leading the CL intensity to de-crease and more active oxygen to produce.

Based on the above results, the main reactioncould be as follows:

caviation .2HzO ) 2'OH + 2H + photons (1)OH + ATX-70~'OH + ATX-70 (2)ATX-70+20z~10z+0z- +ATX-70 (3)In the experiments, another phenomenon was

also found. It is the ATX-70 that can reduce thethreshold of sonoluminescence. Experimentsshowed that when the power was 35 W, sonolumi-nescence began to appear in the liquid withoutATX-70. However, the power decreased to about28 W when ATX-70 was added. So ATX-70 makesthe cavitations become easier. The ATX-70 maybeact as some sort of surface activator to strengthenthe cavitations, just like described by MiyoshPJ.

.0 LumiMl rox-10

+ FeLA

-\ 1 -m TLIT

J:I1nrllJf~tf

'0

11~ .0c:J=E~30

~

jd 00 ~

.0 WrJmr TW...

~ -'A0-/-

0

lDrosoni::

10 zo JO

Time(.)to so

Figure 6. The change of CL intensity for prove the produc-tion of active oxygen. The data was means:!: S. D. (n =3)

4 Conclusion

..

In this paper, the main work is to discuss therole of ATX-70 in the process of cavitations. Theresults showed that the active oxygen, which wasused to kill the tumor cells in SDT, was mainlyproduced by the chemical reaction in the. process ofcavitations instead of coming from cavitations di-rectly. The main functionof ATX-70 in cavitationsis to transfer the energy of . OH to the dissolvedoxygen in the liquid, and turn the oxygen into 10zand 02. Though ATX-70 increases the yield of ac-tive oxygen, its molecular structure does notchange, just like some sort of catalyze. The resultalso showed that ATX-70 is a highly efficient

AcknowledgmentsThanks for the help of Professor Da Xing,

who works in South China Normal University,gives me some useful advices for the discussion.Thanks for the help of Yi Zheng, who gives mesome advices for the writing.

Correspondenceto:Chunfeng DingHenan Key Laboratory of Laser and Optic-electricInformation TechnologyZhengzhou UniversityZhengzhou, Henan 450052, ChinaTelephone: 86-371-6776-7625Email: dingchf@zzu.edu. en

IIII

J

,jJ1J~Ij~J

JI

References1. Jin Z, MiyoshiN, Ishiguro K, et al. Combinationeffect

of photodynamicand sonodynamictherapy on experimen-tal skin squamous cell carcinoma in C3H/HeN mice. JDermato12000; 27 (5): 294-306.

2. KesselD, La J, Jeffers R, et al. Modesof photodynam-ic vs. sonodynamic cytotoxicity. J Photochem PhotobiolB 1995: 28 (3): 219-21.

3. Miyoshi N, Misik V, Riesz P. Sonodynamictoxicity ofgallium-porphyrin analogue ATX-70 in human leukemiacells. RadiatRes1997: 148 (0: 43-7.

4. Worthington AE, Thompson J, Rauth AM, et al.Mechanism of ultrasound enhanced porphyrin cytotoxici-ty. Part I: A search for free radical effects, UltrasoundMed Bioi 1997; 23 (7): 1095 -105.

5. Yumita N, Umemura S, Nishigaki R. Ultrasonicallyin-duced cell damage enhanced by photofrin IT : mechanismof sonodynamicactivation. In Vivo 2000: 14 (3): 425-9.

6. Kessel D, Jeffers R, Fowlkes JB, et al. Effects of sono-dynamic and photodynamic treatment on cellular thiollevels. J Photochem Photobiol B 1996; 32 (1- 2): 103-6.

7. Sakusabe N, Okada K, Sato K, et al. Enhanced sono-dynamic antitumor effect of ultrasound in the presence ofnonsteroidal anti-inflammatory drugs. Jpn J Cancer Res1999; 90 (10): 1146-51.

8. Miyoshi N, Igarashi T, RieszP. Evidenceagainst singletoxygen formation by sonolysisof aqueousoxygen-saturat-ed solutions of Hematoporphyrin and rose bengal. Themechanism of sonodynamictherapy. Ultrason Sonochem2000; 7 (3): 121-4.

9.Hristov PK, Petrov LA, Russanov EM. Lipid peroxida-tion induced by ultrasonication in Ehrlich ascitic tumorcells. Cancer Lett 1997; 121 (0: 7-10.

1O.Misik V, Riesz P. Free radical intermediates in sonody-namic therapy. Ann NY Acad Sci 2000: 899: 335 - 48.

11. Misik V, Miyoshi N, Riesz P. EPR spin trapping studyof the decomposition of azo compounds in aqueous solu-tions by ultrasound: potential for use as sonodynamic sen-sitizers for cell killing. Free Radic Res 1996; 25 (0: 13-22.

I

IJ

,

t

f

i

I.

J

I

l

)I.

J

}

J

I. 88 .

r

~ Life Science Journal, 3 (4), 2006, Ding, et al, Sonosensitization Mechanism of ATX-70

I\

12. Misik V, Riesz P. Peroxyl radical fonnation in aqueoussolutions of N, N-dimethylfonnamide, N-methylfor-mamide, and dimethyl-sulfoxide by ultrasound: implica-tions for sonosensitized cell killing. Free Radic Bioi Med1996; 20 (1): 129-38.

13. Miyoshi N, Misik V, Fukuda M, et al. Effect of galli-um-porphyrin analogue ATX-70 on nitroxide fonnationfrom a cyclic secondary amine by ultrasound: on themechanism of sonodynamic activation. Radiat Res 1995;143 (2): 194 - 202.

14. Nakano M. Detection of active oxygen species in biologi-cal systems. Cellular and Molecular Neurobiology 1998;18 (6): 565 - 79.

15. Matsugo S, Konishi T, Matsuo D, et al. Reevaluationof superoxide scavenging activity of dihydrolipoic acid and

\

~

k~

II.

,"r,~

\

,

"

~

.l

-~

\I

\

l\~

its analogues by chemiluminescent method using 2-methyl-6-( p-methoxyphenyl)-3, 7-dihydroimidazo [1,2-aJ pyrazin-3-one (MCLA) as a superoxide probe.Biochem Biophys Res Comm 1996; 227: 216 - 20.

16.UeharaK, MaruyamaN, HuangCk, etal. The firstapplication of a chemiluminescence probe,2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1, 2-aJ pyrazin-3-one (MCLA), for detecting 02 production, in vitro,from Kuppfer cells stimulated by phorbol myristate ac-etate. FEBS Letters 1993; 335 (2): 167 -70.

17. Tianxi Hu. Free Radical Life Science Progress. AtomicEnergy Publishing Company 1997; 5: 65 -77.

ReceivedJune 17, 2006

I.

. 89 .