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© 2008 by the American Institute of Ultrasound in Medicine
J Ultrasound Med 27:57-64 • 0278-4297

Study of the Synergistic Effect on Hepatoma 22 Tumor Cells by Focused Ultrasound Activation of Hematoporphyrin

Quanhong Liu, PhD, Xiaoying Li, MD, Lina Xiao, MD, Pan Wang, MD, Xiaobing Wang, MD and Yan Wang, MD

College of Life Sciences, Shaanxi Normal University, Xi’an, China.

Address correspondence to Quanhong Liu, PhD, College of Life Sciences, Shaanxi Normal University, Xi’an, 710062 Shaanxi, China., E-mail: lshaof{at}snnu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective. The synergistic effect of ultrasound and drugs on tumor cells is known as sonodynamic therapy (SDT). The purpose of this study was to evaluate the effects of SDT on lipid peroxidation and the activity of antioxidative enzymes in isolated hepatoma 22 (H-22) cells to better understand the bioeffects of SDT. Methods. The viability of cells was evaluated by the Trypan blue dye exclusion test. The morphologic changes of H-22 cells were observed by a scanning electron microscope immediately after treatment. The intracellular reactive oxygen species levels were detected by 2',7'-dichlorofluorescein diacetate. Colorimetry and enzymatic chemical methods were used to measure the lipid peroxidation levels and activities of key antioxidant enzymes (ie, superoxide dismutase, selenium-dependent glutathione peroxidase, and catalase) in H-22 tumor cells. Results. Our experiments indicated that the ultrasonically induced cell damage rate was increased with 100-µg/mL hematoporphyrin, whereas no cell damage was observed with hematoporphyrin alone. Generation of reactive oxygen species in cell suspensions after SDT treatment was remarkably higher than in controls. The malondialdehyde content was remarkably enhanced, and antioxidative enzyme activities were obviously decreased compared with controls. Conclusions. This study suggests that oxygen free radicals may play an important role in improving membrane lipid peroxidation and decreasing the activities of key antioxidant enzymes in cells. It was speculated that this biological mechanism might be involved in mediating the killing effect of H-22 cells in SDT.

Key Words: antioxidative enzymes • hematoporphyrin • hepatoma 22 • lipid peroxidation • reactive oxygen species • sonodynamic therapy

Abbreviations: CAT, catalase • DCFH-DA, 2',7'-dichlorofluorescein diacetate • Hp, hematoporphyrin • H-22, hepatoma 22 • LPO, lipid peroxidation • MDA, malondialdehyde • ROS, reactive oxygen species • SDT, sonodynamic therapy • Se-GSH-Px, selenium-dependent glutathione peroxidase • SEM, scanning electron microscope • SOD, superoxide dismutase • TBA, thiobarbituric acid


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Modern cancer treatment usually involves combinations of different modalities to maximize the therapeutic outcome and to reduce side effects.1 Sonodynamic therapy (SDT), the synergistic effect of ultrasound and chemicals (sonosensitizers) on tumor cells, is a promising modality for cancer treatment.2–4 Ultrasound can be focused in a small region and can penetrate deeply in tissues,5 which then locally activate a preloaded sonosensitizer.6 Although hematoporphyrin (Hp), as a special sonosensitizer (Figure 1Go), showed preferential and long-term retention in tumor tissues compared with normal tissues,7,8 Hp alone did not show any cell-damaging effect. When it was activated by ultrasound, producing singlet oxygen and other free radicals,5,9–11 then the free radicals could cause irreversible damage to tumor cells. This makes it possible to damage the pathologic site with minimal damage to peripheral healthy tissues; therefore, SDT has potential for targeted therapy.


Figure 1
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  		Figure 1. Structure of Hp.

 
Recently, SDT was mainly focused on the mechanisms of killing effects by using different ultrasonic parameters and different sonosensitizers.12 A series of theories have been proposed, such as singlet oxygen,9,13 alkoxyl radicals,13,14 lipid peroxidation (LPO),15 apoptosis,16,17 and so on. However, the mechanism of SDT is influenced by multiple factors, including the nature of the biological model, the sonosensitizer, and the ultrasonic parameters.18 The exact mechanism of SDT is still unknown.

To date, we have done many studies on the killing effects of ultrasound combined with Hp on different tumor cell lines and found that the damage to tumor cells may be related to reactive oxygen species (ROS).17 Kawabata and Umemura19 also reported that sonochemically produced ROS may play an important role in ultrasonically induced cell killing in the presence of Hp, and Yeh et al20 found that the accumulation of ROS might promote LPO and change the antioxidative system. Yumita et al10 suggested that sonodynamically induced LPO in membranes is the primary mechanism of sonodynamically induced hemolysis with Hp. It is well known that ROS are produced during normal aerobic metabolism; an imbalance between the production and detoxification of ROS results in oxidative stress. However, studies related to the antioxidative system in SDT have been scarce until now. Therefore, the aim of this study was to evaluate the effects of SDT on LPO and the activity of antioxidative enzymes in isolated hepatoma 22 (H-22) cells to have a better understanding about the bioeffects of SDT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Hematoporphyrin and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St Louis, MO). Malondialdehyde (MDA), selenium-dependent glutathione peroxidase (Se-GSH-Px), catalase (CAT), and superoxide dismutase (SOD) detection kits were obtained from Nanjing Jiancheng Biotechnology Institute (Nanjing, China). All other reagents were commercial products of analytical grade.

Tumor Cells
The H-22 cells and Institute of Cancer Research mice were supplied by the Experimental Animal Center of Shaanxi, Chinese Traditional Medicine Institution (Xi’an, China). The cell lines were passaged 3 to 5 days through Institute of Cancer Research mice weighing 18 to 22 g in the form of ascites. Hepatoma 22 cells were harvested from the peritoneal cavity of tumor-bearing mice 5 to 7 days after inoculation, suspended in saline collected by centrifugation, and then resuspended in saline at a concentration of 1 x 106 cells/mL. The cell suspensions were stored on ice until needed.

Ultrasound Exposure System
The experimental setup for insonation is shown in Figure 2Go. An ultrasound transducer with a diameter of 38 mm and a focal length of 52 mm was submerged in the bottom of a glass container filled with cold degassed water. A polystyrene sample test tube containing 1.0 mL of the cell suspension was placed into the focal area of the transducer for insonation.


Figure 2
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  		Figure 2. Ultrasonic exposure system.

 
The ultrasound transducer was manufactured by the Institution of Applied Acoustics, Shaanxi Normal University. The same transducer was used for all the experiments, with a resonant frequency of 1.43 MHz in a standing wave mode, and it was used to convert the electrical power measured by the amplifier (T&C Power Conversion, Inc, Rochester, NY) into acoustic power. To specify the intensity in the insonation experiment and to have an easy and obvious understanding, we used the reading output power from the amplifier representing the spatial average ultrasonic intensity in our experimental system. Additionally, our previous investigation indicated that the real in situ intensity in the standing wave mode could be about 60% to 70% of the electrical power. For all experiments, cold degassed water (4°C) was used as the ultrasound coupling medium, thus reducing the thermal effect caused by ultrasound irradiation. The temperature within the cell suspensions was checked with a thermometer, and we found the temperature rise to be unlikely to induce thermal damage of cells during such a short duration.

Detection of Cell Damage
Ultrasonically induced cell damage in the presence and absence of Hp in suspensions was detected by staining the isolated H-22 cells with Trypan blue dye. A 0.2-mL aliquot was taken from the cell suspension immediately after a fixed period of insonation and mixed with 0.2 mL of 0.4% Trypan blue solution. Cell integrity was determined by counting the number of unstained cells on the glass plate of a hemocytometer using an optical microscope. The integrity was checked before every treatment, and cell suspensions with integrity of greater than 99% were used in this series of experiments. This proportion of intact cells before treatment was regarded as the standard for integrity determination after insonation.

Scanning Electron Microscope Observation
Immediately after ultrasound treatment, cells were fixed in 2.5% glutaraldehyde in 0.1-mol/L phosphate-buffered saline (pH 7.2–7.4). Then they were fixed in 1% osmium tetroxide, washed in phosphate-buffered saline, dehydrated by graded alcohol, displaced, dried at the critical point, gold evaporated, and observed with a scanning electron microscope (SEM; Quanta 200, Philips-FEI, Best, the Netherlands)

Measurement of ROS Generation
The rate of DCFH-DA was measured by a fluorescence microplate reader (Zenyth 3100, Anthos Company, Salzburg, Austria). 2',7'-Dichlorofluorescein diacetate is a sensitive and widely used compound for detection of intracellular oxidant production. Oxidation of DCFH-DA creates highly fluorescent dichlorofluorescein.

The DCFH-DA was added to the cell suspension. It diffuses across the cell membrane and is hydrolyzed by intracellular esterases to dichlorofluorescein, which, on oxidation, yields highly fluorescent 2',7'-dichlorofluorescein. The final concentration of DCFH-DA was 10 µmol/L. The samples were incubated at 37°C in the dark for 20 minutes. Fluorescence was measured with the fluorescence microplate reader with excitation of 488 nm and a 530-nm emission filter.

Biochemical Analysis
Products of LPO in cells were estimated by the thiobarbituric acid (TBA) method, according to the directions of an MDA detection kit. This is an improved colorimetric method to determine the content of lipid peroxide by using a Unicam UV300 (Thermo Electron Corporation, Waltham, MA). Malondialdehyde, which is a stable end product of fatty acid peroxidation, reacts with TBA at acidic conditions to form a complex that has maximum absorbance at 532 nm. The optical density was measured at 532 nm for the MDA concentration. The results were expressed as nanomoles per milliliter.

Superoxide dismutase activity was estimated according to the directions of an SOD detection kit. The method is based on the generation of oxygen produced by xanthine and xanthine oxidase, which react with 2-(4-todophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a complex, which has maximum absorbance at 550 nm. Superoxide dismutase activity was measured by the degree of inhibition of this reaction. The results were expressed as units per milliliter.

Selenium-dependent glutathione peroxidase estimation was based on the following principle: Se-GSH-Px catalyzed the degradation of hydrogen peroxide to water at the expense of reduced glutathione. Reduced glutathione could react with 5-sulfurs, replacing 2 nitro group benzoic acids to form a yellow complex, which had maximum absorbance at 412 nm. The Se-GSH-Px activity was measured by monitoring the increase of absorbance at 412 nm. The results were expressed as units per milliliter.

Catalase activity was measured according to the directions of a commercial CAT kit. The method is based on the fact that CAT dismutates hydrogen peroxide into water and oxygen, although the reaction could be inhibited by ammonium molybdate. The residual hydrogen peroxide could react with ammonium molybdate to form a complex. The change in absorbance was observed at 405 nm. The results were expressed as units per milliliter.

Statistical Analysis
The results are presented as mean ± SD from 3 or 4 individual experiments (n = 6–8). Statistical evaluation of the difference in relation to sonicated cells was performed with the Student t test; P < .05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell Damage
The intact fractions of H-22 cells in the presence of 100-µg/mL Hp for up to 60 seconds of exposure at an ultrasonic intensity of 2 W/cm2 are shown in Figure 3Go. There was no apparent difference between the Hp group and the control group. After 60 seconds of exposure, the viability of cells was reduced to 63.7% in the presence of 100-µg/mL Hp but only 87.3% without Hp. The survival rate of cells was lower in the ultrasound group than the control group (P < .05). Compared with the other 3 groups, ultrasonically induced cell damage in the presence of Hp was significantly enhanced (P < .01).


Figure 3
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  		Figure 3. Viability of H-22 cells after different treatments. CT indicates control group with no treatment; Hp, 100-µg/mL Hp alone; US, ultrasound alone; and US+Hp, ultrasound plus 100-µg/mL Hp. Error bars represent SD of the means (n = 6). **P < .01 compared with the ultrasound group; {Delta}{Delta}P < .01 compared with the Hp group; ++P < .01 compared with the control group.

 
Results of SEM Observation
Scanning electron microscope observation of the surface of isolated H-22 cells after 60 seconds of exposure at an ultrasonic intensity of 2 W/cm2 in the presence and absence 100-µg/mL Hp is shown in Figure 4Go. The control group showed untreated cells with numerous microvilli over the surface of the cells (Figure 4AGo); Hp alone had only a slight effect on the surface of the cells and had no visible difference with the control group (Figure 4BGo). Cells irradiated with continuous wave ultrasound (1.43 MHz) showed a significant decrease in the number of microvilli, and several small craters were also seen (Figure 4CGo). Although cells exposed to identical ultrasound conditions in the presence of 100-µg/mL Hp were seriously damaged, with apparent deformation (Figure 4DGo), some papillous protuberances were seen on the surface of cells, where the cytoplasm seemed to have extruded through the membrane boundary.


Figure 4
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  		Figure 4. Scanning electron microscope images of H-22 cells. A, Control group with untreated intact cells. B, Cells with 100-µg/mL Hp alone. C, Cells irradiated with ultrasound alone. D, Cells irradiated with ultrasound in the presence of 100-µg/mL Hp.

 
Level of ROS
Sonodynamic treatment resulted in distinct effects on the level of ROS (Figure 5Go). The results showed that Hp had synergistic effects with ultrasound and significantly increased the ROS content compared with the other 3 groups (P < .01). The level of ROS was significantly increased after ultrasound exposure alone in comparison with the Hp and control groups (P < .01), whereas there were no differences between the Hp and control groups.


Figure 5
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  		Figure 5. Content of ROS in H-22 cells after different treatments. Abbreviations are as in Figure 3Go. Error bars represent SD of the means (n = 6). **P < .01 compared with the ultrasound group; {Delta}{Delta}P < .01 compared with the Hp group; ++P < .01 compared with the control group.

 
Malondialdehyde Concentration and Antioxidative Systems
To evaluate the peroxidation properties of Hp and ultrasound, the spectrophotometry method was used to measure the product of membrane LPO (MDA and other TBA-reactive substances). As seen in Figure 6Go, Hp showed synergistic effects with ultrasound and significantly increased LPO compared with the other 3 groups (P < .01). On the other hand, compared with the control group, ultrasound-induced LPO was found to be statistically evident (P < .05), but no changes in peroxidation were observed after treatment with Hp alone.


Figure 6
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  		Figure 6. Content of MDA in H-22 cells after different treatments. Abbreviations are as in Figure 3Go. Error bars represent SD of the means (n = 6). *P < .05, **P < .01 compared with the ultrasound group; {Delta}{Delta}P < .01 compared with the Hp group; ++P < .01 compared with the control group.

 
Sonodynamic treatment caused a marked effect on the key constituent of the antioxidative defense system (Figure 7Go). According to the results, ultrasound combined with Hp caused a significant decrease in the activities of antioxidative enzymes (SOD, Se-GSH-Px, and CAT) compared with the other 3 groups (P < .01; Figure 7Go). Also, we observed a significant trend for the decrease in the activities of antioxidative enzymes (SOD and Se-GSH-Px) after ultrasound treatment in comparison with the control and Hp groups (P < .05; Figure 7, A and BGo). Furthermore, CAT activity was significantly decreased after ultrasound exposure alone in comparison with the control group (P< .01), but no difference was found between the control and Hp groups (Figure 7CGo).


Figure 7
Figure 7
Figure 7
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  		Figure 7. Antioxidative enzyme activities in H-22 cells after different treatments. A, Activity of SOD. B, Activity of Se-GSH-Px. C, Activity of CAT. Abbreviations are as in Figure 3Go. Error bars represent SD of the means (n = 6). **P < .01 compared with the ultrasound group; {Delta}P < .05, {Delta}{Delta}P < .01 compared with the Hp group; +P < .05, ++P < .01 compared with the control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The synergistic killing effects of ultrasound combined with Hp on isolated H-22 cells were investigated. We found that the cell damage rates with the combination of ultrasound and Hp were more significantly increased than in the other 3 groups. Under the SEM, changes in the cell membrane ultrastructure observed in the experiments were obvious (Figure 4Go). The ultrasonically induced cell membrane damage was much more serious in the presence of Hp than in the presence of ultrasound alone. Sonodynamic treatment caused a marked increase in the level of ROS. Meanwhile, the MDA content with the combination of ultrasound and Hp was remarkably enhanced. In addition, the activities of the antioxidative enzymes (SOD, Se-GSH-Px, and CAT) with the combination of ultrasound and Hp were evidently decreased compared with the other 3 groups. It can thus be hypothesized that SDT, by elevating the MDA concentration and lowering antioxidative enzyme activities, leads to increased cell damage. Malondialdehyde is one of the end products of LPO, and the MDA content indirectly reflected the degree of ROS, which attacked the cell membrane, whereas the antioxidative enzyme activities indirectly reflected the capacity of the cells, which eliminated ROS. It was speculated that the damage to the tumor cells might have been related to ROS.

Reactive oxygen species such as the superoxide anion, hydrogen peroxide, the superoxide radical, and the hydroxyl radical are often internally formed as products of normal metabolism. However, endogenously overproduced ROS spontaneously attack proteins, lipids, nucleic acids, and other biologically important molecules and thus damage the cells.21 Malondialdehyde, which is produced by the oxidation of polyunsaturated fatty acids in membranes induced by ROS, is an indicator of oxidative damage.22 Our experiments revealed that the levels of ROS and MDA with the combination of ultrasound and Hp were more remarkably enhanced compared with the other 3 groups. It was indicated that ROS may play an important role in ultrasonically induced cell damage. At the same time, the SEM showed that the cell membrane ultrastructure was seriously damaged with the combination of ultrasound and Hp. Our findings are in agreement with earlier studies. Our previous study17 showed that there was visibly increased LPO in tumor cells after SDT treatment. Yumita et al10 pointed out that sonodynamically induced LPO with Hp was able to cause erythrocyte lysis. In our experimental model, cell membrane changes could be reflected clearly by LPO and morphologic observation under the SEM. The experiments implied that the ultrasonically induced cell damage may be induced by LPO products to modify the physiologic properties of the cell membrane,23 thus causing membrane depolarization, disturbing the asymmetry of membrane lipids, inducing inhibition of membrane enzymes that modulate transport of proteins, causing a loss of plasmatic membrane integrity,22 and so on.

To control the overproduction of ROS, animals have developed a complex antioxidative defense system including SOD, Se-GSH-Px, CAT, and other enzymes.24 Superoxide dismutase catalyzes the dismutation of superoxide anions to hydrogen peroxide.25 Catalase catalyzes the degradation of hydrogen peroxide to water and oxygen. Selenium-dependent glutathione peroxidase catalyzes the reduction of hydrogen peroxide to water at the expense of reduced glutathione. It can also remove organic hydroperoxides.26 Those antioxidative enzymes can eliminate excessive generation of ROS to keep the balance between the production and detoxification of ROS. Our experiments indicated that the depletion of antioxidative enzyme activities (SOD, Se-GSH-Px, and CAT) with the combination of ultrasound and Hp was much more significant than in the other 3 groups. The consequence of changes in key antioxidative enzymes may lead to an increase of ROS formation and oxidative stress induction. The excessive generation of ROS can result in substantially higher LPO at the cellular and molecular levels,27 finally causing oxidative damage.

In conclusion, we have shown the effects of SDT on LPO and antioxidative enzyme activities in isolated H-22 cells. The observed damages to the cellular membrane and changes in antioxidative enzyme activities may induce cell death. It was speculated that the synergistic cell-killing effect observed during sonolysis of cells in the presence of Hp may have been due to ROS that were generated by the sonosensitiser.28–33 On the one hand, ROS attacked the lecithoid polyunsaturated fatty acids of the cell membrane, causing LPO, which affected the membrane structure and function and eventually led to cell damage; on the other hand, ROS interacted with the antioxidative defense system, causing denaturalization of the antioxidative enzymes and finally bringing on cell death. However, the mechanism of SDT is influenced by multiple factors; thus further investigations are needed.


   Footnotes
 
Received July 2, 2007, from the College of Life Sciences, Shaanxi Normal University, Xi’an, China. Revision requested July 23, 2007. Revised manuscript accepted for publication September 13, 2007.

This work was supported by National Natural Science Foundation of China grants 39870240 and 30270383 and the Excellent Doctor Innovation Project of Shaanxi Normal University.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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