Ultrasonics Sonochemistry 31 (2016) 437–448

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Ultrasonics Sonochemistry

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Sinoporphyrin sodium triggered sono-photodynamic effects on breastcancer both in vitro and in vivo

http://dx.doi.org/10.1016/j.ultsonch.2016.01.0381350-4177/� 2016 Published by Elsevier B.V.

⇑ Corresponding author at: Key Laboratory of Medicinal Resources and NaturalPharmaceutical Chemistry, Ministry of Education, National Engineering Laboratoryfor Resource Developing of Endangered Chinese Crude Drugs in Northwest of China,College of Life Sciences, Shaanxi Normal University, Xi’an 710119, Shaanxi, China.

E-mail address: wangxiaobing@snnu.edu.cn (X. Wang).

Yichen Liu, Pan Wang, Quanhong Liu, Xiaobing Wang ⇑Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of EndangeredChinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, Shaanxi, China

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

Article history:Received 6 October 2015Received in revised form 23 January 2016Accepted 29 January 2016Available online 29 January 2016

Keywords:DVDMSSono-photodynamic therapyAntitumorMetastasis

Sono-photodynamic therapy (SPDT) is a promising anti-cancer strategy. Briefly, SPDT combinesultrasound and light to activate sensitizers that produce mechanical, sonochemical and photochemicalactivities. Sinoporphyrin sodium (DVDMS) is a newly identified sensitizer that shows great potential inboth sonodynamic therapy (SDT) and photodynamic therapy (PDT). In this study, we primarily evaluatedthe combined effects of SDT and PDT by using DVDMS on breast cancer both in vitro and in vivo. In vitro,DVDMS-SPDT elicits much serious cytotoxicity compared with either SDT or PDT alone by MTT and col-ony formation assays. 20 ,70-Dichlorodihydrofluo-rescein-diacetate (DCFH-DA) and dihydroethidium(DHE) staining revealed that intracellular reactive oxygen species (ROS) were significantly increased ingroups given combined therapy. Terephthalic acid (TA) method and FD500-uptake assay reflected thatcavitational effects and cell membrane permeability changes after ultrasound irradiation were alsoinvolved in the enhancement of combination therapy. In vivo, DVDMS-SPDT markedly inhibits the tumorvolume and tumor weight growth. Hematoxylin-eosin staining and immunohistochemistry analysisshow DVDMS-SPDT greatly suppressed tumor proliferation. Further, DVDMS-SPDT significantly inhibitstumor lung metastasis in the highly metastatic 4T1 mouse xenograft model, which is consistent well withthe in vitro findings evaluated by transwell assay. Moreover, DVDMS-SPDT did not produces obviouseffect on body weight and major organs in 4T1 xenograft model. The results suggest that by combinationSDT and PDT, the sensitizer DVDMS would produce much better therapeutic effects, and DVDMS-SPDTmay be a potential strategy against highly metastatic breast cancer.

� 2016 Published by Elsevier B.V.

1. Introduction

Breast cancer is the leading malignancy among women in theworldwide [1]. Traditional therapies such as surgery, radiotherapy,chemotherapy, have made great achievements in clinic controlling,but accompanied by severe side effects. Therefore, efficient andnovel therapies that reduce the high mortality rate and improvepatient quality of life are urgent.

Photodynamic therapy (PDT), proposed by Dougherty et al., is aclinically approved modality for cancer treatment, and firstapproved by the FDA for certain kinds of cancer in 1998 [2,3]. Sincethe 1990s, PDT has been gradually applied for treatment of variouscancers because it is a non-invasive, highly localized, and relativelysafe form of therapy [3–6]. However, PDT application is limited to

superficial lesions as the limited penetration of laser light [7].Inspired by PDT, Yumita used ultrasound to activate sensitizers,which was named sonodynamic therapy (SDT) [8,9]. The major dif-ference between SDT and PDT is the energy source used to activatethe sensitizer. Compared with laser light used in PDT, SDT usesultrasound that can easily penetrate deep tissue layers where somemalignancies reside, thereby makes up the major limitation of PDT[9–13].

Sono-photodynamic therapy (SPDT) is an emerging approach inthe anti-cancer field using the combination of SDT and PDT. Thebasis of this therapy is to administer a small amount of sensitizer,which can be activated by both ultrasound and light simultane-ously to produce mechanical, sonochemical and photochemicalactivities [14]. Previous studies have indicated that SPDT canpotentiate notable antineoplastic activity against a variety ofmalignancies at pre-clinical and clinical levels [15–18]. Someresearchers reported that the combined therapy produced moreobvious anti-cancer effect than any monotherapy, and furtherdecreased the dosage of sensitizer and the energy of ultrasound

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or light [17,18]. Wang et al. report three cases of advancedrefractory breast cancer by SPDT treatment, the three patientshad proven metastatic breast carcinoma and failed to respond toconventional therapy but had significant partial or completeresponses after treated with SPDT [19]. Kenyon et al. show thatthe combined therapy can significantly extend predicted mediansurvival time and improve quality of life for patients [20]. Collec-tively, the above theoretical and clinical researches demonstrateSPDT is of worthy further investigation as an alternative strategyfor conventional therapy.

Although the PDT mechanism involving type I and type II reac-tions has been clearly illustrated [21], the mechanisms of SDT aresomewhat difficult to explain. In view of the physical propertiesof ultrasound, various factors such as ROS formation, thermaleffect, mechanical effect and cavitational effect are possiblyinvolved in the enhancement of therapeutic effect [22]. While,the combination of SDT and PDT, SPDT refers to several key factors,such as the parameters of ultrasound and light, the sensitizers, theresponse of tumors, the sequence of ultrasound and light exposure,etc.

Nevertheless, sensitizers are of great importance in SPDT pre-clinical and clinical trials [14]. Until now, a sensitizer Sonnelux-1-SPDT has treated hundreds of cases with different types oftumors, in which breast cancers showed higher response to SPDT[19,20]. Our recent studies show the sensitizer Sinoporphyrinsodium (also referred to DVDMS) could be highly activated by bothlight and ultrasound [23–29]. This study aims to explore the pre-clinical possibilities of DVDMS-SPDT, additionally, the underlyingmechanisms such as radical generation, mechanical effect trig-gered alteration of membrane permeability, and differentsequences of light and ultrasound exposure are also carefullyanalyzed.

In this study, the anti-proliferation and metastatic inhibitionelicited by DVDMS-mediated SPDT was investigated on breast can-cer cells both in vivo and in vitro. The findings may provide impor-tant implications for DVDMS application in the treatment ofcancer.

2. Materials and methods

2.1. Sensitizers

Sinoporphyrin sodium (DVDMS) was kindly provided by Profes-sor Qicheng Fang from the Chinese Academy of Medical Sciences(Beijing, China). It has a purity of 98.5%. DVDMS was dissolved ina physiological saline solution to a final storage concentration of1 mM and was stored in the dark at �20 �C. The chemical structureof DVDMS is shown in Fig. 1.

Fig. 1. The chemical structure of DVDMS.

2.2. Reagents

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bro-mide tetrazolium (MTT), paraformaldehyde, Triton X-100, bovineserum albumin (BSA), crystal violet, terephthalic acid (TA), fluores-cein isothiocyanate-dextran (FD500) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 20,70-Dichlorodihydrofluo-rescein-diacetate (DCFH-DA) was from Molecular Probes Inc. (Eugene, OR,USA). Dihydroethidium (DHE) was purchased from Invitrogen(Thermo Scientific Inc., US). Primary antibody against proliferatingcell nuclear antigen (PCNA) was purchased from Abcam(Cambridge, UK). Secondary antibodies were obtained from ZhongShan Golden Bridge Biotechnology (Beijing, China).

2.3. Tumor cell lines

Mouse mammary cancer 4T1 cell line was obtained from thedepartment of basic medicine, Union Medical College, Beijing,China. Human breast cancer cell lines MDA-MB-231 and MCF-7were obtained from the cell bank of the Chinese Academy ofScience, Shanghai, China. The cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM, Gibco, Life Technologies, Inc.,USA) supplemented with 10% fetal bovine serum (Hyclone, USA),100 U/ml penicillin, 100 lg/ml streptomycin and 1 mM

L-glutamine, in an incubator with 5% CO2 and 100% humidity at37 �C. Cells in the exponential phase of growth were used in eachexperiment.

2.4. Animals

The BALB/c mice (female, 18–20 g body weight) were suppliedby the Experimental Animal Center of Fourth Military MedicalUniversity (FMMU) (Xi’an, China) and housed at room temperaturewith a 12 h light/dark cycle and allowed free access to food andwater. After 1 week’s acclimation, BALB/c mice weresubcutaneously injected at the right flanks with 0.1 ml 4T1 cells(1 � 107 cells/ml). When the tumor reached a size of 60–70 mm3,the tumor-bearing mice were randomly assigned to differentgroups and ready for experiment. All animal experiments werecarried out with the approval of the university’s institutionalanimal care and use committee.

2.5. In vitro laser light and ultrasound treatment

Cells in the exponential phase were collected and re-suspendedin a complete culture medium at cell densities of 2 � 105 cells/mlin 35 mm cell culture dish (Corning Inc. Tewksbury MA, USA) for12 h. Then all samples were randomly divided into differentgroups: control group (Control), ultrasound group (US), 0.5 lMDVDMS group (DVDMS), DVDMS plus ultrasound group (SDT),DVDMS plus laser light group (PDT), DVDMS plus ultrasound pluslaser light group (SPDT) and DVDMS plus laser light plus ultra-sound group (PSDT). For all groups except the control and ultra-sound, cells were incubated with 0.5 lM DVDMS for 3 h,allowing sufficient time for cells to uptake the sensitizer rapidlyto achieve a maximum level. It is worth mentioning that, becausethe breast cancer 4T1, MDA-MB-231 and MCF-7 cells are adherent-dependent growth, in order to investigate the natural biologicalresponse to ultrasound treatment, we exposed cells to ultrasoundand light irradiation when they were normal adherent growthwithout any trypsinization.

For laser light, a semiconductor laser (excitation wavelength:635 nm; manufacturer: Institute of Photonics & Photon Technol-ogy, Department of Physics, Northwest University, Shaanxi, China)was used as previous report [25]. Laser irradiance was measured

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using a radiometer system (Institute of Photonics & Photon Tech-nology, Department of Physics, Northwest University). As forin vitro experiments, the laser was used with a power intensityof 23 mW/cm2 and an irradiation time of 60 s such that the finaldose of light is 1.4 J/cm2.

For the in vitro ultrasound set-up [30]. The planar ultrasoundapparatus (Fig. 2), manufactured by Sheng Xiang High TechnologyCo., LTD (China), was applied in this study. Ultrasonic frequency ofthis apparatus was 0.84 MHz. The diameter of its planar transducerwas 35 mm. The ultrasound intensity was calibrated before it wasdisplayed in the LED screen of the apparatus. An intensity of0.25 W/cm2 and duration of 60 s was used for ultrasound treat-ment. For irradiation, the interval between transducer and cell cul-ture plate was filled with ultrasound couplant to facilitateultrasound transmission. Temperature increase inside the cultureplates was measured before and after ultrasound treatment witha digital thermometer, and no significant variation of temperaturewas detected (±1 �C). Thus, any bio-effects observed in this studywere considered to be non-thermal.

2.6. Cell viability assays

Cell viability was evaluated using conventional MTT assay andcolony formation assay. For MTT assay, cytotoxicity was detectedat 4 h after different treatments as set forth [26]. Cellviability was calculated as follows equation: Cell survival (%) =ODtreatment group/ODcontrol group � 100%.

Moreover, the colony-formation assay was adopted forexamination of long-term proliferative potential in this study. Afterdifferent treatment, cells were seeded onto 35 mm culture dishesat a density of 1000 cells/well, and subsequent protocols wereperformed as previous described [25]. Then the total number ofcolonies was counted and proliferation potential was calculatedusing the following equation: relative colony formation rate (%)= number of colonies with at least 50 cells in the treatmentgroup/number of colonies with at least 50 cells in the controlgroup � 100%. The experiment was conducted in triplicate.Representative views were also photographed.

2.7. Cell motility

Cell motility was evaluated using transwell assays as previouslyreported [25]. 100 ll 2.5 � 105 cells/ml were seeded into the topchamber of a Corning chamber (Corning Pharmingen, San Diego,CA) in serum-free medium containing 0.3% BSA. Medium contain-ing 10% serum was placed in the lower chamber. After 20 h, cellsthat migrated to the underside of the membrane were detectedusing a 0.1% crystal violet solution. The assay was repeated threetimes with three replicates each. Cells that migrated to the under-side of the membrane were quantified using light microscopy(Nikon Eclipse TE2000-S, Japan). Crystal violet was dissolved usinga 33% acetic acid, and the OD ratio at 570 nm was determinedusing a microplate reader.

Fig. 2. Diagram of ultrasound setup. An ultrasound transducer is in diameter of 35 mm. Bculture dishes containing cells suspension was exposed above the transducer. The frequ

2.8. Determination of intracellular ROS and superoxide anion

Intracellular ROS level was assessed by measuring the fluores-cence intensity of dichlorofluorescein (DCF) as described in ourprevious article [31]. Briefly, cells were incubated with 10 lMDCHF-DA at 37 �C for 30 min prior to laser light and/or ultrasoundtreatment. At 2 h after treatment, the cells were washed inphosphate-buffered saline and then imaged using an E-600 fluo-rescence microscope (Nikon Corporation, Tokyo, Japan). MultilabelReader was used to further validate the intensity of DCF. The treat-ment was the same with above. At 2 h after treatment, cells wereharvested and lysed in 100 ll phosphate-buffered saline. Fluores-cence of the supernatant was measured on a Multilabel Reader(PE EnSpire, USA) at an excitation wavelength 488 nm withemission wavelength at 500–550 nm.

Dihydroethidium (DHE), a selective probe for superoxide anion(O2

��), was used to further identify intracellular ROS [32]. Briefly,cells were incubated with 5 lM DHE at 37 �C for 30 min prior tolaser light and/or ultrasound treatment. At 2 h after treatment,the samples were washed in phosphate-buffered saline andharvested by trypsinization, then detected immediately by flowcytometry (Guava easyCyte 8HT, Millipore, Billerica, MA).

2.9. Evaluation of ultrasound caviation

Acoustic cavitation of ultrasound was evaluated using TA(terephthalic acid) method. When the acoustic power is above athreshold in a liquid system, some active species such as hydroxylradicals (OH�) and hydrogen peroxide (H2O2) are formed on ther-molysis of H2O during ultrasound exposure [33]. TA solution(1 mM) as dosimeter solution reacts with a hydroxyl radicalformed during ultrasound irradiation (0.5-, 1.0- and 1.6 W/cm2

for 60 s), forms 2-hydroxyterephthalic acid (HTA) which aredetected using fluorescence photometer (LS-55, PE, USA) at426 nm.

2.10. Detection of cell membrane permeability

In order to measure the changes of membrane permeabilityinduced by ultrasound used in SPDT/PSDT, FD500-uptake assaywas performed. FD500 is the conjugate of fluorescein FITC and dex-tran with a molecular weight of 500,000 that cannot freely pene-trate the cell membrane. Once membrane permeability isenhanced due to some stress, such as sonication, FD500 may enterthe cells, subsequently, the intracellular fluorescent signal could bedetected [18]. 4T1 cells were sonicated in the presence of 1 mg/mlof FD500. After ultrasound irradiation (0.25-, 0.5-, 0.75- and1W/cm2), cells were immediately washed with PBS, then theFD500-positive cells were quantified by flow cytometry.

2.11. In vivo sonodynamic and photodynamic therapy

For in vivo experiments, the laser light treatment equipmentwas same as the in vitro one. The laser was utilized with a power

etween transducer and cell culture plate was filled with ultrasound couplant. 35 mmency of the ultrasound is 0.84 MHz.

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intensity of 417 mW/cm2 and an irradiation time of 2 min suchthat the final dose of light is 50 J/cm2. For the in vivo ultrasoundtreatment, because previously we found the tumor tissuesresponded well to the focused ultrasound with a frequency of1.90 MHz [27]. Here, we would like to use this focused ultrasoundto treat tumors. As described in previous paper, the focused ultra-sound transducer with a frequency of 1.90 MHz, manufactured bythe Institution of Applied Acoustics, Shaanxi Normal University(Xi’an, China), was submerged in degassed water in the tank facingdirectly upward. A continuous sine-wave signal was generated andamplified by a multifunctional generator (AG1020; T&C PowerConversion Inc., Rochester, New York, USA) before feeding thetransducer. An average intensity of 1.6 W/cm2 (ISATA) was usedfor ultrasound treatment for 3 min. For the experiments, colddegassed water (4 �C) was used as the ultrasound coupling med-ium, thus reducing hyperthermia during treatment. The tempera-ture close to the exposed region showed no significant variation(<3 �C).

The tumor-bearing mice were randomly divided into fivegroups: Control, PDT, SDT, SPDT and PSDT. The control mice didnot receive any treatment. The other four groups of mice wereinjected with DVDMS (2 mg/kg) via the caudal vein, and after24 h post injection, they were exposed to laser light (PDT), ultra-sound (SDT), or ultrasound immediately followed by laser light(SPDT) or laser light immediately followed by ultrasound (PSDT).Prior to laser light and ultrasound applications, hair from thetumor region was removed using depilatory cream. The mice wereanesthetized with 1% pentobarbital sodium and placed on a platefor PDT treatment or on a plexiglass plate with degassed waterfor SDT treatment. For SDT, the naked tumor region was immersedinto the degassed water and the center of the tumor was exposedto the focused ultrasound spot for 3 min. The overview protocol isshown in Fig 3.

The therapeutic results of each group were evaluated by mea-suring the tumor volumes for 12 days, and the body weight wasalso measured. The long (a) and short (b) diameters of the tumors

Fig. 3. Diagram of in vivo DVDMS-SPDT treatment protocol. Tumor-bearing mice were randid not receive any treatment. The other four groups of mice were injected with DVDMlaser light (PDT), ultrasound (SDT), or ultrasound immediately followed by laser light (SPwith a power intensity of 417 mW/cm2 and an irradiation time of 2 min such that the fiMice were exposed to ultrasound for 1.6 W/cm2, 3 min in this study.

were measured using slide calipers every day after treatment. Themean tumor volume was calculated using the formula: ab2/2, andthe volume inhibition ratio was calculated as follows: (1-averagetumor volume of treated group/average tumor volume of the con-trol group) � 100%. 12 days after treatment, the mice weresacrificed, and the tumors were removed and weighed. In addition,the lungs were removed and fixed in Bouin’s solution for 24 h. Thepulmonary nodules were photographed and counted.

2.12. Hematoxylin and eosin (H&E) straining

Tumors and major organs was fixed using 10% formalin for atleast 24 h. Samples were then paraffin-embedded, sectioned, andstained with H&E. Histopathological changes were observed usinglight microscopy (Nikon E600, Japan).

2.13. Immunohistochemistry

Paraffin-embedded tumor tissue sections (7 lm thick) weredewaxed, rehydrated, and then treated with heat-mediated anti-gen retrieval using 10 mM citrate buffer (pH 6.0) for 15 min.Sections were permeabilized with 0.2% Triton X-100 for 15 min.Sections were immersed in 3% hydrogen peroxide solution for10 min to quench endogenous peroxidase activity. Non-specificbinding was prevented by incubation with 5% normal goat serumfor 15 min. The sections were then incubated with anti-PCNA anti-body overnight at 4 �C. Antibody binding was detected usinghorseradish peroxidase-conjugated secondary antibody for20 min at 37 �C. Sections were visualized using diaminobenzidine(DAB) solution counterstained with hematoxylin, and observedusing light microscopy.

2.14. Statistical analysis

Quantitative data in all statistical analyses was set asmean ± standard deviation of three samples obtained from three

domly divided into five groups: Control, PDT, SDT, SPDT and PSDT. The control miceS (2 mg/kg) via the caudal vein, and after 24 h post injection, they were exposed toDT) or laser light immediately followed by ultrasound (PSDT). The laser was utilizednal dose of light is 50 J/cm2. The in vivo frequency of the ultrasound was 1.90 MHz.

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independent experiments, and the statistical significance wasdetermined using one-way analysis of variance (ANOVA), dataare represented as p-values <0.05, <0.01, <0.001 were consideredstatistically significant.

3. Results

3.1. Cytotoxicity assessment

Cytotoxicity of DVDMS-mediated SDT and/or PDT in vitro wasevaluated on 4T1, MDA-MB-231 and MCF-7 cell lines by MTT assay24 h after different treatments. As shown in Fig. 4A–C, DVDMSalone (0.5 lM) and ultrasound alone groups did not show obviouscytotoxicity on all breast cancer cell lines. Compared with control,two single treatment increased the cytotoxicity of 4T1, MDA-MB-231 and MCF-7 cells to 27.36%, 34.88% and 24.44% for SDT, and36.69%, 40.16% and 38.58% for PDT respectively (p < 0.01 vs. con-trol). When treated by the combination of SDT and PDT, markedlyloss in cell viability was observed among tree breast cancer celllines. Therein, the cell viability loss of 4T1, MDA-MB-231 andMCF-7 cells after treated with SPDT was 80.49%, 81.69% and70.69%, respectively. After PSDT treatment, the viability loss ofthe above cell lines was 85.01%, 86.13% and 77.48% respectively.These data indicate a synergistic enhancement of cytotoxicitywhen compared with SDT alone (p < 0.001) or PDT alone(p < 0.001).

A colony formation assay on 4T1 cells was further confirmedthe reduced proliferative potential induced by the combination ofSDT and PDT. As shown in Fig. 5, numerous colony formed in con-trol and DVDMS alone groups had no effect on colony formationability. In contrast, colony formation ability of 4T1 cells in SPDTand PSDT groups decreased significantly stronger than that inSDT and PDT treatment alone. The colony formation rate in DVDMSalone, SDT, PDT, SPDT, PSDT groups were 99.02%, 68.78% (p < 0.01vs. control), 59.51% (p < 0.01 vs. control), 12.68% (p < 0.001 vs. con-trol, p < 0.001 vs. SDT/PDT), 8.29%, respectively (p < 0.001 vs. con-trol, p < 0.001 vs. SDT/PDT). This result is consistent well with theMTT assay above.

3.2. DVDMS mediated SPDT/PSDT inhibits cell migration in 4T1 cells

Since metastasis is the primary cause of mortality in breast can-cer patient, we evaluated the effect of cell migratory ability treatedby DVDMS-SPDT on cell migration using a transwell assay [34].Numerous cells in the control group migrated to the underside ofthe well at 20 h post treatment, while a significant decrease of cellmigration was observed in PDT alone. When compared with SDTand PDT alone, the combined therapies further decreased the rateof cell migration to a lower level (Fig. 6). Analysis of the OD ratio ofcrystal violet indicated similar results. The findings showed 44.29%

Fig. 4. In vitro cytotoxicity assessment of DVDMS-SPDT on 4T1, MDA-MB-231 and MCindependent experiments. **p < 0.01 versus control, ***p < 0.001 versus control, ###p < 0.0group.

(p < 0.01), 59.94% (p < 0.01) decreases in cell migration rate in SDTand PDT groups with 0.5 lM DVDMS exposure, respectively. Whilea markedly decrease with 87.57% (p < 0.001 vs. control), and91.59% (p < 0.001 vs. control) was seen in the SPDT and PSDTgroups at the same drug dose.

3.3. DVDMS-mediated combination treatment (SPDT/PSDT) drasticallyenhance the ROS production

Cell damage induced by PDT or SDT is closely related to the gen-eration of intracellular ROS [25,31]. We examined whether combi-nation treatment can enhance the ROS production in 4T1 cells.Therefore, intracellular ROS level was measured using DCFH-DAstaining and DHE staining after SDT and/or PDT treatment. As indi-cated in Fig. 7, compared with control, PDT increased ROS genera-tion for about 50 times (p < 0.01), whereas SDT showed little DCFfluorescence intensity enhancement for 15 times per milligramprotein. Importantly, SPDT and PSDT treatments dramaticallyenhanced intracellular ROS levels, which were about 180–240times higher compared with control (p < 0.001) and were about4–5 times higher compared with SDT and PDT (p < 0.01). DHEstaining (Fig. 8) showed that, compared with the control, SDTand PDT caused 19.2% (p < 0.05) and 27.5% (p < 0.05) high DHE flu-orescence. Under the same conditions, much high DHE fluores-cence were observed when cells were treated with SPDT andPSDT, in which the high DHE fluorescence increased to 55.3%(p < 0.01) and 62.15% (p < 0.01), respectively.

3.4. Ultrasound-induced cavitational effect

Acoustic cavitation is a complicated process that leads to bothmechanical shear stress and free radicals formation arising fromthe collapse of oscillating bubbles [35]. In our study, TA (tereph-thalic acid) method was used to detect the degree of cavitationcaused by ultrasound treatment. Fig. 9 shows that at 1.9 MHzfocused ultrasound treatment, HTA fluorescence intensityincreased slightly after 0.5 W/cm2 (p < 0.05) and increased dramat-ically after 1- and 1.6 W/cm2 (p < 0.01) ultrasound treatment,which indirectly implies the utilized parameters would causecavitational effect.

3.5. Ultrasound-induced membrane permeability

Cell membrane integrity induced by ultrasound used in ourstudy was evaluated by FD500-uptake assay to demonstratedmechanical effects of ultrasound. FD500 does not stick on the cellmembrane, and rarely enters the dead cells [18]. Thus, the uptakeof FD500 can be a credible sign to reflect the membrane permeabil-ity [18]. As shown in Fig. 10, proportions of 4T1 cells exhibitinghigh fluorescence intensities of FD500 increased to 7.22%, 25.42%,

F-7 cells were determined by MTT assay. Error bars represent the SD from three01 SPDT/PSDT group versus SDT group, &&&p < 0.001 SPDT/PSDT group versus PDT

Fig. 5. In vitro cytotoxicity assessment of DVDMS-SPDT on 4T1 cells determined by colony formation assay. Error bars represent the SD from three independent experiments.**p < 0.01 versus control, ***p < 0.001 versus control, ###p < 0.001 SPDT/PSDT group versus SDT group, &&&p < 0.001 SPDT/PSDT group versus PDT group.

Fig. 6. Analyses of change in cell migration on 4T1 cells using a transwell assay. Cells in each group move to the lower surface of them filter were stained with crystal violetand photographed under a light microscope (a–e). a: Control, b: SDT, c: PDT, d: SPDT, e: PSDT. The OD ratio of crystal violet were measured (bottom right corner). Error barsrepresent the SD from three independent experiments. **p < 0.01 versus control, ***p < 0.001 versus control, ###p < 0.001 SPDT/PSDT versus SDT, &&&p < 0.001 SPDT/PSDTversus PDT.

Fig. 7. Intracellular ROS generation in 4T1 cells at 2 h post treatment. The cells were labeled with DCFH-DA and observed by fluorescence microscope (Left side). a: Control, b:DVDMS alone, c: SDT, d: PDT, e: SPDT, f: PSDT. The mean fluorescence intensity of the oxidized product DCF in cells was detected by Multifunctional microplate reader (Rightside).

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Fig. 8. Intracellular superoxide anion generation in 4T1 cells at 2 h post treatment. Cells were labeled with DHE and detected by flow cytometry.

Fig. 9. Evaluation of ultrasound caviation by TA method. The mean HTA fluores-cence was detected after different intensity of ultrasound irradiation for 60 s usingfluorescence photometer at 426 nm. Error bars represent the SD from threeindependent experiments. *p < 0.05, **p < 0.01 versus control.

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34.50% and 45.62% after 0.25-, 0.5-, 0.75- and 1W/cm2 planarultrasound treatment, respectively. These results confirm thatultrasound process mode used in this study could cause injury ofcell membrane with an ultrasonic intensity dependent mannerindicating the mechanical effects involved in SPDT/PSDT as wellas SDT in our study.

3.6. DVDMS mediated SPDT/PSDT enhance inhibition effect on tumorgrowth

To investigate the in vivo anti-cancer efficacy of combinationtreatment, a 4T1 mouse mammary cancer model was utilized.Tumor-bearing mice were divided into five experimental groups,and result was shown in Fig. 11. Representative mice were also

photographed at 12 days after the corresponding treatments(Fig. 11A). DVDMS mediated SDT/PDT markedly inhibited tumorgrowth (volume and weight) (Fig. 11B–D), and the effect wasnoticeably enhanced with the combination of these two method.The tumor weight inhibiting ratios in the SDT and PDT groups were46.66% and 55.54% (p < 0.01 vs. control). Importantly, for SPDT andPSDT, the tumor weight inhibition ratio was 81.45% and 84.83%(p < 0.001 vs. control, p < 0.01 vs. SDT/PDT), indicating that DVDMSmediated SDT and PDT repressed tumor growth and that the effectwas strengthened when the two therapy combined.

3.7. DVDMS mediated SPDT/PSDT inhibits lung metastasis in vivo

Mice were sacrificed and assessed for the extent of metastasisto the lungs by examining the gross appearance of pulmonary nod-ules after fixation with Bouin’s solution for 24 h. Lungs of mice inthe control group displayed multiple metastasized tumors of vari-ous sizes on their surface (Fig. 12). In contrast, the surfaces of thelungs from both SPDT and PSDT treated mice, which were verysimilar to the normal lung. While either single treatment of SDTand PDT reduced lung metastases partly. The pulmonary noduleswere manually counted and the average numbers of Control,SDT, PDT, SPDT, PSDT groups were 22 ± 2.38, 12 ± 2.22 (p < 0.001vs. control), 10.2 ± 2.18 (p < 0.001 vs. control), 2 ± 1.21 (p < 0.001vs. control, p < 0.01 vs. SDT/PDT), 1.6 ± 1.26 (p < 0.001 vs. control,p < 0.01 vs. SDT/PDT), respectively.

3.8. Histological and immunohistochemical analysis of in vivo anti-tumor effects

For histopathological analysis using H&E staining (Fig. 13), weobserved that the tumor tissue from the control group displayedcompact tumor cells with an intact structure. In the SDT or PDTgroups, the tumor tissues were no longer structurally integrated:there were sections devoid of cells and there were numerousnuclear fragments. The tissue damage was more severely extensive

Fig. 10. Detection of cell membrane permeability by FD500-uptake assay. 4T1 cells were sonicated in the presence of 1 mg/ml of FD500 conjugated with fluorescein FITC andthen the FD500-positive cells were quantified by flow cytometry.

Fig. 11. DVDMS-SPDT inhibits tumor growth. (A) Representative photos of 4T1 tumor-bearing mice from different groups at day 12 after treatment. (B) Tumor weight ofdifferent groups of tumor-bearing mice after treatment. (C) Image of tumors excised from each group of mice 12 days after treatment. (D) Tumor growth curves of differentgroups of tumor-bearing mice after treatment. Error bars represent the SD from three independent experiments. **p < 0.01 versus control, ***p < 0.001 versus control,##p < 0.01 SPDT/PSDT group versus SDT group, &&p < 0.01 SPDT/PSDT group versus PDT group.

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Fig. 12. DVDMS-SPDT inhibits lung metastasis in vivo. (A) Photos of lungs after soaking in Bouin’s solution showing spontaneous pulmonary breast cancer metastases (blackarrows and dotted circle). (B) The pulmonary nodules were manually counted and the average numbers were calculated in different groups. **p < 0.01 versus control,***p < 0.001 versus control, ###p < 0.001 SPDT/PSDT group versus SDT group, &&&p < 0.001 SPDT/PSDT group versus PDT group.

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in the SPDT and PSDT group compared with either single treatedgroup.

To determine the effect of combination on cell proliferationin vivo, immunohistochemistry analysis was performed to evaluatethe expression level of PCNA, a representative marker of prolifera-tion [36]. PCNA was highly expressed in the control group and hada reduced expression level in the SDT and PDT groups; the levelwas further greatly decreased in the SPDT and PSDT groups thaneither single treatment group (Fig. 14).

3.9. Evaluation of side effects using DVDMS mediated SPDT/PSDT

The potential in vivo toxicity of DVDMS mediated combinationtreatment of SDT and PDT was also preliminary examined. Wedid not detect any overt signs of toxic side effects or changes inbody weight with drug dose at 2 mg/kg (Fig. 15A). Furthermore,we harvested the major organs including the heart, liver, spleen,and kidney. We were unable to detect any organ damage usingH&E staining (Fig. 15B).

4. Discussion

Sono-photodynamic therapy (SPDT) combines ultrasonic irradi-ation and laser light with drugs known as sensitizers that amplifyits ability to inflict preferential damage on malignant cells [16].Both preliminarily pre-clinical and clinical studies have indicatedthat the combined therapy can overcome major limitations ofPDT and enhance the deep-preferentially ability with selectivelytargeting of the tumor tissue [15,16,19,20]. Ultimately, the com-bined therapy performed stronger anti-cancer effect than any

Fig. 13. Tumor sections were stained with hematoxylin and eosin (HE) after different

monotherapy and may decrease the sensitizer dosage and ultra-sound or light energy, which can further reduce the side effectscaused by the sensitizer [20–22].

Sensitizer is key component in both PDT and SDT. DVDMS is akind of porphyrins photosensitizer isolated from Photofrin II. Com-pare with Photofrin II, DVDMS has advantages of 98.5% chemicalpurity, high water solubility, good targeting and brief skin sensitiv-ity [24]. Previous work have shown that DVDMS preformed greatactivities in both SDT and PDT treatment, which indicated thatDVDMS may be a great candidate sensitizer in SPDT treatment[25,27–29]. Therefore, we agog to assess the anti-cancer effect ofDVDMS-SPDT, and investigate whether DVDMS-SPDT can performa markedly combination effect.

Herein, we used the BALB/c-derived mouse mammary carci-noma cell line 4T1, which share numerous characteristics withhuman mammary carcinoma, including immunogenicity, growthcharacteristics, and metastatic properties, to investigate the effectsof sono-photodynamic therapy on cell metastasis and proliferationboth in vivo and in vitro.

Firstly, we investigate the tumor inhibition effect of DVDMS-SPDT both in vitro and in vivo. For in vitro studies, we preliminarilyassess the cytotoxicity of DVDMS-mediated SPDT in 4T1, MDA-MB-231 and MCF-7 cell lines. A significant cell viability loss (Fig. 4A–C)was found in combined treatment groups with presence of 0.5 lMDVDMS 24 h after treatment in each cell line. Colony formationassay on 4T1 cells support the conclusion from the point of long-term proliferative ability (Fig. 5). The two assays confirm that com-bination treatment evoked by DVDMS is more efficiency than SDT/PDT alone at the same DVDMS dose. Moreover, in in vivo study,tumor volume and weight in SPDT and PSDT groups were signifi-cantly inhibited in comparison with those in each single treatment

treatments. Histopathological changes were observed under a light microscope.

Fig. 14. Immunohistochemistry detection for PCNA after different treatments.

Fig. 15. Evaluation of side effects using DVDMS-SPDT. (A) A plot of body weight versus the number of days post different treatments. (B) Effect of different treatments on thestructural changes of major organs in 4T1-bearing mice. Major organ sections were stained with hematoxylin and eosin (H&E). Histopathological changes were observedunder a light microscope. Data were shown as mean ± SD from eight mice in each group.

446 Y. Liu et al. / Ultrasonics Sonochemistry 31 (2016) 437–448

group (Fig. 11A–D). H&E staining showed obvious tissue destruc-tion with large spaces post SPDT and PSDT treatment (Fig. 13). Fur-ther, the expression level of PCNA were significantly downregulated in SPDT and PSDT groups compared with control, SDTand PDT groups (Fig. 14). Besides, comparing with our previouswork on DVDMS-PDT, despite promising results used 150 J/cm2

on the treatment of same xenograft mode, SPDT using the lightdose of 50 J/cm2 with a more efficiency treatment effect are evensafer than conventional PDT [25]. One probable explanation is thatthe increased treatment efficiency may owe to subsequent SDTcould compensate for the unavoidable attenuation PDT effect onthe deeper tissues.

As tumor metastasis is one of the major challenges in cancertreatment, in this study, we also focused on metastatic ability of4T1 cells after different treatments [34]. Transwell assay demon-strated that SDT and PDT treatment could mildly suppress cellmigration, whereas SPDT and PSDT can almost completely inhibitcell migration 20 h after treatment (Fig. 6). Furthermore, as breastcancer cells usually metastasize to lungs [37,38]. The lung metas-tasis assay was performed to assess the metastatic ability after dif-ferent treatments. Results in Fig. 12 demonstrated that the numberof pulmonary nodules in the PSDT and SPDT groups weresignificantly less than that in control and single treatment groups,indicating that the combined treatments greatly inhibited themetastasis of tumor cells to the lungs. This result is consistent wellwith the in vitro findings, suggesting that DVDMS-SPDT canefficiently suppress migration and metastasis in 4T1 cell bothin vitro and in vivo.

Above results confirm that DVDMS-SPDT can achieve bettertherapeutic results on inhibition effect of tumor proliferation andmetastasis compared with either single treatment, that was similarto what have been found in previous studies on SPDT [19,20],

which indicating that the newly identified sensitizer, DVDMS,can be potentially used in SPDT treatment.

Next, it is necessary to explore the possible underling mecha-nisms on enhancement of cell killing by the combination. It is wellknown that oxidation stress is one of the key factors in the tumordestruction of PDT and might be also involved in mechanism ofSDT [39]. Excessive ROS can damage lipids, proteins, and DNA aswell as cause mitochondrial dysfunction, deregulate ion balance,and cause loss of membrane integrity [31]. Therefore, the ROS levelwas assessed in order to explore whether the production of ROSplays a crucial role in anti-cancer effect of DVDMS-SPDT. The result(Figs. 7 and 8) suggested a markedly increased oxidative stress in4T1 cell responded to DVDMS-SPDT, which might provide strongcontribution to the enhanced anti-cancer effect of the combinationtreatment.

Although oxidative stress has been proved to be involved inmechanisms of SDT and PDT, unlike light, due to the physical prop-erties of ultrasound, various mechanisms other than ROS such asthermal effect, mechanical effect and cavitational effect are possi-bly responsible for the enhancement of therapeutic effect [9,40].Acoustic cavitation is a complicated process that leads to bothmechanical shear stress and free radicals formation arising fromthe collapse of oscillating bubbles. The degree of bio-effectsdepends on the given ultrasound intensity and frequency and theother experimental conditions [40]. In this study, TA method wasused to detect the degree of cavitation caused by ultrasound treat-ment. Result in Fig. 9 confirms that the present parameter cancause acoustic cavitation and may induce a series of chemical reac-tions and mechanic shearing, ultimately resulting in enhancementof cell damage. Meanwhile, multiple reports have indicated thatultrasound can increase the permeability of the plasma membrane[41,42]. In this paper, we use FD500-uptake assay to measure the

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changes of membrane permeability induced by ultrasound. Thedata suggest that ultrasound used in in-vitro study can cause per-meability alteration of cell membrane with an ultrasonic intensitydependent manner (Fig. 10). The increased permeability of cellmembrane may further induce improvement of drug uptake, andwork to delivery of sensitizer into tumor cell, which possibly causemore efficiency anticancer effect [43].

Moreover, we evaluated the effect of combined therapy withdifferent treatment orders and interestingly found that when PDTwas performed before SDT, the combination effect was relativelystronger than that in case of SDT prior to PDT. Wang et al. andJin et al. (from two independent groups) reported similar resultsin their study [16,39]. Based on these results, we tentatively inter-pret this to the combination of PDT with subsequent SDT has a firstchoice of PDT dependent on oxygen concentration followed bySDT, which is not completely dependent of oxygen. Finally, duringthe whole treatment period, there were no obvious side effects,such as significant body weight drop and detectable internalorgans damage (Fig. 15A and B), which indicating the relativesafety of this combined treatment.

5. Conclusions

In this study, DVDMS mediated SPDT produce stronger thera-peutic effects on breast cancer over SDT and PDT alone bothin vitro and in vivo and DVDMS-SPDT may be a potential strategyagainst highly metastatic breast cancer. The excess level of ROSmay plays a crucial role in cell death, cavitational effect andchanges of membrane permeability induced by ultrasound alsowork on the enhancement of combination therapy. Due to thecomplicated system of SPDT, the mechanism becomes more elu-sive and there is no certain theory about SPDT. Thereby, more indepth preclinical studies and clinical trials are certainly neededto verify and prefect this new method.

Acknowledgements

This work was supported by National Natural Science Founda-tion of China (Grant Nos. 81472846, 81571834) and the Funda-mental Research Funds for the Central Universities (Grant No.GK201502009).

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