Ultrasonics 50 (2010) 634–638

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Sonodynamic effects of protoporphyrin IX disodium salt on Ehrlich ascetictumor cells

Pan Wang, Lina Xiao, Xiaobing Wang, Xiaoying Li, Quanhong Liu *

Key Laboratory of Medicinal Plant Resources and Natural Pharmaceutical Chemistry, College of Life Sciences, Shaanxi Normal University, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 February 2008Received in revised form 1 February 2010Accepted 2 February 2010Available online 6 February 2010

Keywords:Cell damageEhrlich ascetic tumor cellsProtoporphyrin IX disodium saltUltrasound

0041-624X/$ - see front matter � 2010 Elsevier B.V.doi:10.1016/j.ultras.2010.02.001

* Corresponding author. Address: College of LifeUniversity, Xi’an, Shaanxi 710062, China. Tel.: +86 29

E-mail address: lshaof@snnu.edu.cn (Q. Liu).

The cytotoxic effect of protoporphyrin IX disodium salt (PPIX) on isolated Ehrlich ascetic tumor (EAT)cells induced by ultrasound exposure was investigated. Tumor cells suspended in air-saturated phos-phate buffer solution (PBS, pH 7.2) were exposed to ultrasound at 2.2 MHz for up to 60 s in the presenceand absence of PPIX. The viability of cells was determined by a trypan blue exclusion test. The morpho-logical changes of cells in SDT were observed by scanning electron microscope (SEM). And the sub-cellu-lar localization of PPIX in EAT cells was detected by confocal laser scanning microscopy (CLSM). Theultrasonically-induced cell damage increased as PPIX concentration increased, while no cell damagewas observed with PPIX alone. CLSM observation revealed that the fluorescence of PPIX and rhodamine123 (mitochondrial probe) overlapped very well in the cytoplasm. The results indicate that PPIX couldenhance the ultrasonically-induced cell damage and mitochondria may play an important role duringsonodynamically induced cytotoxicity.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

Sonodynamic therapy (SDT) is a promising new modality forcancer therapy based on the synergistic effect on cell killing bythe combination of a sonosensitizer and ultrasound [1,2]. Sonosen-sitizers such as porphyrin show preferential accumulation andretention in tumor tissues compared with in the normal tissue[2]. Ultrasound is a mechanical wave that could penetrate tissueand maintain the ability to focus energy into target tissue [3]. Aseries of experiments in vivo and in vitro presented that the poten-tial applications of sonodynamic action for cancer therapy [4,5].However, the exact mechanism of the sonodynamic effect remainscontroversial. Yumita et al. [6] suggested that singlet oxygen pro-duced during the sonodynamic effect of porphyrins is responsiblefor the reported cytotoxicity. While some studies proposed thatthe sonochemically produced peroxyl and alkoxyl radicals maybe the primary reason for ultrasonically-induced cell damage[7,8]. Kinoshita and Hynynen [9] reported that hyperthermiacaused by ultrasound may play an important role in the sonody-namic effect induced by porphyrin.

In the current studies, the majority of sonosensitizers are por-phyrins and their derivatives [5,6], but other compounds, such aschemotherapeutic drugs and anti-inflammatory drugs have alsobeen tested for sonodynamic activity [10,11]. Protoporphyrin IX

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Sciences, Shaanxi Normal85310275.

(PPIX) is known to be a hematoporphyrin derivative. The chemicalstructure is shown in Fig. 1. PPIX involves in heme biosynthesisand can be accumulated in tumor cells or tissues not only byadministration PPIX itself but also by administration its precursorssuch as 5-aminolevulinic acid (5-ALA). The cause is that the levelsof ferrochelatase to metabolize PPIX to heme decreased in prolifer-ating tissues [12,13]. ALA-induced PPIX mediated photodynamiceffects were studied in the past decades [14–17]. But there arefew reports about PPIX induced sonodynamic effects. Kinoshitaand Hynynen [9] reported that PPIX could be considered as aneffective sonosensitizer in SDT. Umemura et al. [18] suggested thatPPIX has more significant sonodynamic effect than hematoporphy-rin (Hp). In our previous studies, it was showed that PPIX could en-hance the ultrasonically induced sarcoma 180 cells killing in vivoand in vitro [19,20]. And it is confirmed that PPIX might have morepotential cytotoxicity than Hp when irradiated with ultrasound[20]. In this study, we use Ehrlich ascetic tumor (EAT) cells asmaterial to investigate the sonodynamic effect of PPIX, in orderto have a better understanding about PPIX as an effective sonosen-sitizer in sonodynamic treatment.

2. Materials and methods

2.1. Chemicals

Protoporphyrin IX disodium salt (PPIX) and rhodamine 123 werepurchased from the Sigma chemical company (St. Louis, MO, USA).All other reagents were commercial products of analytical grade.

Fig. 1. Structure of protoporphyrin IX.

P. Wang et al. / Ultrasonics 50 (2010) 634–638 635

2.2. Tumor cells preparation

Ehrlich ascetic tumor (EAT) cells and Institute of Cancer Re-search (ICR) mice were supplied by the Experimental Animal Centerof Shaanxi, Chinese Traditional Medicine Institution. The cell lineswere passaged weekly through ICR mice weighing 18–22 g in theform of ascites. EAT cells were harvested from the peritoneal cavityof tumor-bearing mice 7–10 days after inoculation, suspended in anair saturated phosphate buffer solution (PBS, pH 7.2), collected bycentrifugation, then re-suspended in PBS at a concentration of1.0 � 106 cells/ml. The cell suspensions were stored on ice untilneeded. Before every treatment, the cell viability was checked toensure that cell integrity was above 99% in a series of experiments.The experiment animals were treated according to the standardssupported by the animal protection committee of China.

2.3. Ultrasound exposure system

The experimental set-up for insonation was similar as previ-ously described (Fig. 2) [19]. The ultrasound transducer was man-ufactured by the Institution of Applied Acoustics, Shaanxi NormalUniversity (Xi’an, China). This transducer with a diameter of15 mm and a focal length of 22 mm was submerged in the bottomof a glass container filled with cold degassed water. A polystyrenesample test tube containing 1.0 ml cell suspension was placed intothe focal area of the transducer for insonation. The same trans-ducer was used for all the experiment, with a frequency of2.2 MHz in a standing wave mode, and it was used to convertthe electrical power measured by the amplifier (T&C Power Con-version, Inc., Rochester, New York, USA) into acoustic power. In or-der to specify the intensity in the insonation experiment and havean easy and obvious understanding, we usually use the readingoutput power from the amplifier representing our spatial averageultrasound intensity in our experiment system. For all experi-ments, cold degassed water (4 �C) was used as the ultrasonic cou-pling medium, thus reducing thermal effect caused by ultrasound

Fig. 2. Ultrasonic exposure equipment.

irradiation. The temperature within the cell suspensions waschecked with a thermometer and found the temperature rise tobe unlikely to induce thermal damage of cells during such shortperiod of duration.

2.4. Cell damage

Immediately after treatment, the evaluation of cell damage wasperformed by trypan blue exclusion test. Total volume of 200 ll ofcell suspension was mixed with an equal amount of 0.4% trypanblue solution in PBS (pH 7.2). Then the number of cells excludingtrypan blue (unstained) was counted using a hemocytometer toestimate the number of intact viable cells and the number ofnon-viable cells. The count before exposure is considered to beabove 99%.

2.5. Scanning electron microscope observation

Immediately after ultrasound treatment, cells were fixed in 2.5%glutaraldehyde in 0.1 M PBS (pH 7.2), then fixed in 1% osmium tet-raoxide, washed by PBS, dehydrated by graded alcohol, displaced,dried at the critical point, gold evaporated, and observed using ascanning electron microscope (ProPalette 7000, Hitachi, Tokyo,Japan).

2.6. Confocal laser scanning microscope observation

Cells were incubated in the dark at 37 �C with 80 lM PPIX forhalf an hour, then rinsed in PBS three times and stained with10 lM rhodamine 123 (Mitochondrial probe) for half an hour at37 �C. Prior to visualization, excess probe was washed off and livecells were submerged in PBS. Image analysis was accomplishedwith the confocal laser scanning microscope (TCS SP5, Leica, Mann-heim, Germany), which is able to create confocal images dependingon a pinhole set assembled in the microscope. A pinhole size of 80–120 lm was used, which provided resolution on the z-axis at about0.2 lm. The excitation and emission wave of PPIX is listed as405 nm and 625 nm, respectively [21]. And the excitation andemission wave of rhodamine 123 is listed as 488 nm and530 nm, respectively [22]. All parameters were kept constant toensure reliable comparison throughout the experiment.

2.7. Statistics

Data were expressed as mean ± standard deviation (SD). Statis-tical analyses were performed using Student’s t-test with a signif-icant level of p < 0.05.

3. Results

3.1. Cell damage

The survival of EAT cells in the absence of PPIX after exposure isshown in Fig. 3. No cell damage was observed in the untreated con-trol group (0 W/cm2). The rate of ultrasonically-induced cell damage(60 s exposure) increased as the ultrasound intensity increased.Compared with control, cell damage was enhanced significantlywhen ultrasound intensity was up to 3 W/cm2 (p < 0.05). Therefore,the intensity threshold for ultrasonically-induced cell damage wasobserved to be around 3 W/cm2. The cell damage was enhancedmarkedly with ultrasound intensity above this threshold.

The intact fractions of EAT cells after exposure as a function ofPPIX concentration at an ultrasonic intensity of 3 W/cm2 is shownin Fig. 4. The ultrasonically-induced cell damage was enhanced asPPIX concentration increased. Under the same ultrasound exposure

0

20

40

60

80

100

0 1 2 3 4 5

Ultrasound intensity (W/cm2)

Cel

l Sur

viva

l%

Fig. 3. Effects of 60 s ultrasonic exposure on EAT cells. The ultrasonic intensity waschanged from 1 to 5 W/cm2 and the ultrasonic exposure time was 60 s. The valueswere obtained from six individual experiments as mean ± SD. Significant effects(p < 0.05) relative to sonicated cells were obtained for untreated control cells.

Fig. 4. The viability of EAT cells after ultrasonic exposure in the presence of PPIX.Ultrasound was used at the intensity 3 W/cm2, and the exposure time was changedfrom 15 to 60 s. The PPIX concentration was changed from 40 to 160 lM. The valueswere obtained from six individual experiments as mean ± SD.

Fig. 5. Effects of ultrasound alone on EAT cells with and without PPIX. Ultrasoundwas used at the intensity 3 W/cm2 and the exposure time was changed from 15 to60 s. The PPIX concentration was 80 lM. The values were obtained from sixindividual experiments as mean ± SD.

636 P. Wang et al. / Ultrasonics 50 (2010) 634–638

conditions, there was no obvious difference between the ultra-sound alone group and ultrasound plus 40 lM PPIX (p > 0.05).But the ultrasonically-induced cell damage was enhanced obvi-ously at 80 lM PPIX (p < 0.05), and became more and more signif-icant as the concentration increased. So the PPIX concentrationthreshold for ultrasonically-induced cell damage was observed tobe 80 lM.

The viability of EAT cells after exposure as a function of expo-sure time at an ultrasound intensity of 3 W/cm2 in the presenceof 80 lM PPIX is shown in Fig. 5. The ultrasonically-induced celldamage was enhanced as the ultrasound exposure time increasedin the ultrasound alone and ultrasound plus PPIX groups. We alsoobserved the enhancement was most significant in the ultrasoundplus PPIX group when the exposure time was 45 s (p < 0.0001)compared with ultrasound alone group. And no significant differ-ences between PPIX alone group and untreated control group werefound (p > 0.05).

3.2. Results of scanning electron microscope observation

The surface of EAT cells after 45 s exposure at an ultrasonicintensity of 3 W/cm2 in the presence and absence 80 lM PPIX is

shown in Fig. 6. Cells in the control group had rich microvilli overthe surface of the cell. PPIX alone had no significant difference withthe control group. The number of microvilli was sharply decreasedwhen cells were treated with ultrasound. While cells were exposedto 3 W/cm2 in the presence of 80 lM PPIX, the morphology of cellschanged seriously.

3.3. Results of confocal laser scanning microscope observation

Rhodamine 123 was used as a molecular probe. It preferentiallylocalizes in undamaged mitochondria of living cells [23] largely be-cause of electrophoretic forces generated by the proton gradientacross the mitochondrial inner membrane [24]. Confocal micro-graphs of the double stained cells with PPIX and rhodamine 123are presented in Fig. 7. In treated cells, diffuse red fluorescencefrom PPIX and green fluorescence from rhodamine 123 wereclearly seen in the cytoplasm.

4. Discussion

Previously, PPIX mediated sensitization of S180 cells to ultra-sound has been well studied in vivo and in vitro experiments.The synergistic effect of PPIX and ultrasound on inducing cell dam-age effect was increasingly apparent at longer exposure lengths.Changes in the cell ultra-structure such as cell membrane destruc-tion, mitochondria swelling and chromatin condensation might beimportant factors that inhibited the tumor growth and even in-duced cell death [19,20].

It is known that the effects of SDT are dependent mainly on theconcentration of porphyrins accumulated in the tumor and theintensity of ultrasound applied [25]. The ultrasonically-inducedcell damage was enhanced as the ultrasonic intensity increased(Fig. 3), and the enhancement became more obvious with theultrasonic intensity above 3 W/cm2. Moreover, we observed theultrasonically-induced cell damage in the presence of PPIX at ultra-sonic intensity 3 W/cm2 (Fig. 4). The results showed that PPIXcould enhance the ultrasonically-induced cell damage, and becamemore and more noticeable as the PPIX concentration increased.When the concentration of PPIX reached 80 lM, ultrasound com-bined with PPIX resulted in more significant cell damage thandid the ultrasound alone under the same experimental conditions.But no cell damage was observed with PPIX alone. On the otherhand, cell damage became more and more significant as the ultra-sound treatment time increased in ultrasound alone and ultra-

Fig. 6. Scanning electron microscope images of EAT cells: (A) control group with untreated intact cells; (B) cells with 80 lM PPIX alone; (C) cells irradiated with ultrasoundalone; (D) cells irradiated with ultrasound in the presence of 80 lM PPIX. Ultrasound was used at the intensity 3 W/cm2 and PPIX at concentration 80 lM.

Fig. 7. Confocal laser scanning microscope images of PPIX and rhodamine 123 loaded cells. Cells were incubated with PPIX (80 lM) for half an hour, washed with PBS andfollowed by 30 min rhodamine 123 incubation. (A, D) rhodamine 123 localization in cells; (B, E) PPIX localization in cells; (C, F) combined image of cells from A, B and D, E,respectively.

P. Wang et al. / Ultrasonics 50 (2010) 634–638 637

sound combined with PPIX groups. Ultrasound plus PPIX groupshowed more marked cell killing effect than ultrasound alone

group at exposure time 45 s (Fig. 5). Under the scanning electronmicroscope, the morphological structure of cells was observed

638 P. Wang et al. / Ultrasonics 50 (2010) 634–638

when irradiated with 3 W/cm2. The ultrasonically-induced celldamage was much more serious in the presence of 80 lM PPIXthan ultrasound alone. Changes in the morphology of cell affect cellfunction, and eventually lead to cell death.

In the past, some data clearly demonstrated that the enhance-ment of the ultrasound induced S180 cells damage by PPIX wasdue to active oxygen generated by ultrasound activating PPIX[19]. Due to the short half-life of active oxygen (approximately4 ls) and the migration distance of active oxygen in biological sys-tems was only about 0.1 lm, the sub-cellular localization of sono-sensitizer is expected to be related to the probable primary sites ofinitial damage by SDT [26–28]. In an attempt to assess the localiza-tion of the PPIX, confocal laser scanning microscope was performedand rhodamine 123 was used as the mitochondrial probe. Rhoda-mine 123 is a useful biological probe because of its high fluores-cence efficiency [29], relatively low toxicity when used at lowconcentration for brief periods, and selective affinity for mitochon-dria of living cells, in particular carcinoma cells [22,26]. Confocallaser scanning microscope detection for both the PPIX and rhoda-mine 123 on the same cell images revealed that both drugs accu-mulated diffusely in the cytoplasm and that mitochondrion is atarget organelle. It was previously shown that PPIX targeted mito-chondria membrane in hamster fibroblasts cells [30]. It was alsoreported that ALA-induced PPIX was initially localized in the mito-chondria, whereas exogenous PPIX was mainly distributed in cellmembranes by means of fluorescence microscopy [28]. Perhapsthe sub-cellular localization of sonosensitizer was dependent onthe chemical structure, the concentration and incubation time ofsonosensitizer and cell type. The sonosensitizer and ultrasoundare known as indispensible determinants for SDT. Additionally,increasing evidence has emerged that the effectiveness of SDTmay also dependent on sub-cellular and intracellular localizationpatterns of a sonosensitizer. This may be related to the fact thatsome organelles of cell and structural elements of tissues are moresensitive than others for cell survival and tissue repair [28]. The re-sults revealed that mitochondria are important binding sites of EATcells in the course of PPIX. We speculate that mitochondria play acentral role in ultrasound induced cell death enhanced by PPIX. It isin agreement with some reports [31–33]. The changes of mito-chondria and the cellular events leading to cell death deserve fur-ther investigation in this model. Other studies have shown that cellmembrane is the most likely site of action for sonochemical effectson cells exposed to ultrasound [19,34]. So plasma membrane maybe another target for SDT. Moreover, it is now recognized thatdamage to certain sub-cellular structure may be correlated to aspecific cell death mechanism.

In conclusion, the results suggest that the synergy between PPIXand ultrasound lead to cell death in EAT cells. It is speculated thatmitochondria may play an important role in the ultrasound com-bined with PPIX induced cell death. But further studies are neededto explain the relation of mitochondria and cell death in this exper-iment model.

Acknowledgments

This work was supported by the Natural Science Foundation forthe Youth (No. 10904087) and the Excellent Doctor Innovation Pro-ject of Shaanxi Normal University.

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