Photochemistry and Photobiology, 2014, 90: 1404–1412

Antitumor Effect of Sinoporphyrin Sodium-Mediated Photodynamic Therapy on Human Esophageal Cancer Eca-109 Cells Jianmin Hu1, Xiaobing Wang1, Quanhong Liu1, Kun Zhang1, Wenli Xiong1, Chuanshan Xu2, Pan Wang*1,2 and Albert Wingnang Leung*2 1

Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, China 2 School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China Received 5 May 2014, accepted 13 August 2014, DOI: 10.1111/php.12333

ABSTRACT The aim of this study was to evaluate the photodynamic effect of Sinoporphyrin sodium (DVDMS). In this study, Eca-109 cells were treated with DVDMS (5 lg mL
1) and subjected to photodynamic therapy (PDT). The uptake and subcellular localization of DVDMS were monitored by flow cytometry and confocal microscopy. The phototoxicity of DVDMS was studied by MTT assay. The morphological changes were observed by scanning electron microscopy (SEM). DNA damage, reactive oxygen species (ROS) generation and mitochondria membrane potential (MMP) changes were analyzed by flow cytometry. Studies demonstrated maximal uptake of DVDMS occurred within 3 h, with a mitochondrial subcellular localization. MTT assays displayed that DVDMS could be effectively activated by light and the phototoxicity was much higher than photofrin under the same conditions. In addition, SEM observation indicated that cells were seriously damaged after PDT treatment. Furthermore, activation of DVDMS resulted in significant increases in ROS production. The generated ROS played an important role in the phototoxicity of DVDMS. DVDMS-mediated PDT (DVDMSPDT) also induced DNA damage and MMP loss. It is demonstrated that DVDMS-mediated PDT is an effective approach on cell proliferation inhibition of Eca-109 cells.

INTRODUCTION Malignant tumors are a leading cause of death worldwide, and the major treatments are focused on surgery, radiotherapy and chemotherapy. However, most of these treatments have inevitable side-effects. So the development of novel treatment strategies is indispensable. Photodynamic therapy (PDT) is a well-established clinical treatment modality for the treatments of tumors. It is based on preferential uptake of photosensitizers in tumor cells or tissues, and subsequent activation with appropriate wavelength of laser light, resulting in tumor cells killing by activated oxygen produced by the photosensitizers (1). As a safe and minimally invasive therapy, it has been clinically applied to many kinds of *Corresponding authors emails: [email protected] (Pan Wang), awnleung@ cuhk.edu.hk (Albert Wingnang Leung) © 2014 The American Society of Photobiology

cancers, such as lung (2), cervical (3), early esophageal (4), bladder (5), melanoma (6) and head and neck cancers (7), etc. Photosensitizers are a critical factor in PDT. Hematoporphyrin derivative (HpD), a complex mixture of porphyrins derived from hematoporphyrin, has been used for localization and photodynamic therapy of tumors (8–10). Numerous studies have been published dealing with the photosensitizing and selective tumordestructive properties of it. These studies have approached the problems from various perspectives: spectroscopic analysis (11), basic cellular properties (12), chemical analysis (13) and animal/ human treatment (14–16). Many studies also focused on its tumor-localizing components (17–21). This consists of proposed chemical structure, photosensitizing properties, biological and biophysical properties et al. Furthermore, Photofrinâ, the commercially available drug enriched in the tumor-localizing fraction of HpD (22–24), which had been approved by the FDA as a photosensitizer in PDT of cancer treatments, is the most widely used photosensitizer so far. Photofrinâ has been approved for clinical use against early- and late-stage lung cancer, esophageal cancer, bladder cancer and malignant, nonmalignant early-stage cervical cancer in the worldwide (25). The study of photofrin can be traced back to 1983 when Dougherty first reported the preparation of photofrin from hematoporphyrin dihydrochloride (26). Since then, many studies have been published dealing with the components of photofrin (27,28). There is general consensus that photofrin is a mixture of dimers and oligomers of hematoporphyrin in which porphyrin units are linked by ether, ester and C–C bonds (25). This indicated that porphyrin dimers or oligomers are possible photosensitizers in photodynamic therapy. And the dimers and oligomers have been the subject of discussion for some time (29,30). To gain further chemical insight into the nature of photofrin or seek for new photosensitizers, many investigators have synthesized dimeric or trimeric porphyrin photosensitizers (31–36). Pandey et al. have synthesized certain porphyrin dimers with ester or ether linkages and tested their photosensitizing activity (31–33). The dimers were found to have better or slighter tumoricidal activity than photofrin. However, a major drawback is that the esters or ethers were found to be unstable at room temperature either as solids or in solution and were also detected hydrolysis in the injection solution within a few minutes of preparation in the Tween 80 aqueous medium used for injection which limited

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Photochemistry and Photobiology, 2014, 90 the application of these agents in clinical PDT. So the study of photosensitizers is ongoing. It would clearly be advantageous to utilize a purified and defined composition in the therapeutic method rather than a complex mixture. However, the active ingredient of photofrin has not been separated into single chemical entity. A drawback is that a long clearance time of 4–8 weeks after injection is required to avoid skin photosensitization (25). In full prescribing information of Photofrinâ, the patients are warned to avoid exposure of skin and eyes to direct sunlight or bright indoor light for at least 30 days following injection with photofrin. These deficiencies also evoked great interest of Professor Qicheng Fang in Chinese Academy of Medical Sciences. He and his colleagues carried out a large number of studies to explore the active fraction of photofrin. The results showed that the fraction which had strong anticancer activities is porphyrin dimer salt connected by an ether bond (It was named Sinoporphyrin sodium, also called DVDMS). This was in accordance with the study of that the active fraction of photofrin is mainly porphyrin dimers or oligomers (26–28). And based on the literatures (31– 36), they synthesized DVDMS and studied the photodynamic activities. The invention had gained China’s intellectual property rights (37). In addition, the applicants evaluated the skin phototoxicity of DVDMS and inhibitory effects of DVDMS-based PDT on the growth of tumors. They found that DVDMS have better tumoricidal activity than photofrin in several tumor-bearing mouse models and with reduced skin phototoxicity than Hiporfin, which had been approved by the China’s State Food and Drug Administration (SFDA) in PDT of cancer treatments (37,38). Wang et al. (39) reported that DVDMS showed higher fluorescence intensity and singlet oxygen production efficiency compared with other photosensitizers (hematoporphyrin, protoporphyrin IX and photofrin) both in cancer cell and normal cell which may have advantages in further clinical applications. Furthermore, DVDMS was also found to have a good targeting to tumor cells or tissues (39–41). The preferential accumulation paves the way for targeted therapy. In addition, DVDMS has better water-solubility, stability and a known chemical structure. All these showed that DVDMS has great potential in clinical PDT. While DVDMS may be a relative ideal porphyrin dimer photosensitizer compared with current photosensitizers, the study of DVDMS-based PDT is not sufficient (only a patent (37) and one literature report (38) can be found). We also think that the general and basic study on it is of vital importance for its clinical application. Therefore, in this study, human esophageal cancer Eca-109 cells were chosen as cell model and we focused on the antitumor effect and primary mechanism of DVDMS-PDT. The results of our study provide optimal parameters and possible mechanisms for DVDMS-mediated PDT, which will be useful for clinical applications.

MATERIALS AND METHODS Sensitizers. Sinoporphyrin sodium (DVDMS) is the product of Qinglong Hi-tech Co., Ltd (Jiangxi, China) and was kindly provided by Professor Qicheng Fang in Chinese Academy of Medical Sciences (Beijing, China). It has a purity of 98.5%. It was dissolved in physiological saline with a storage concentration of 1.25 mg mL
1, and was stored in the dark at
20°C. Its chemical structure is shown in Fig. 1. Photofrin was a gift from Professor Qicheng Fang.

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Figure 1. The chemical structure of DVDMS.

Reagents. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide tetrazolium (MTT), N-acetylcysteine (NAC), propidium iodide (PI) and rhodamine 123 (Rho123) were purchased from the Sigma Chemical Company (St. Louis, MO). 20 ,70 -Dichlorodihydrofluo-rescein-diacetate (DCFH-DA) and Mito Tracker Green (MTG) were supplied by Molecular Probes Inc. (Eugene, OR). All other reagents were commercial products of analytical grade. Cell culture. Human esophageal cancer cell line Eca-109 was obtained from the cell bank of Chinese Academy of Sciences, Shanghai. It was cultured in RPMI-1640 medium (Gibco; Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U mL
1 penicillin, 100 lg mL
1 streptomycin and 1 mM L-glutamine. Cells were maintained at 37°C in humidified 5% CO2 atmosphere. Cells in the exponential phase of growth were used in each experiment. Photodynamic treatment. The semiconductor laser (excitation wavelength: 630 nm; manufacturer: Institute of Photonics & Photon Technology, Department of Physics, Northwest University, Shaanxi, China) was used as a source for evocation of the photodynamic effect. Irradiance was measured by the radiometer system (Institute of Photonics & Photon Technology, Department of Physics, Northwest University). For the laser light, the power intensity: 43.0 mW cm
2; irradiation time: 2–6 min. So the final light dose was varied from 5.2 to 15.6 J cm
2 in this experiment. Cellular uptake of DVDMS detection by flow cytometry. The cells (2 9 105 cells mL
1) were incubated with DVDMS (5 lg mL
1) at 37°C in 24-well culture plates (Corning Inc., New York, NY) for different time intervals. For determining the intracellular DVDMS quantum, cells were collected after different incubation time points (0, 0.5, 1, 2, 3, 4, 5, 6 h) and detected with flow cytometry (Guava easyCyte 8HT; Millipore). The mean fluorescence intensity of DVDMS was recorded at the same measurement conditions. Subcellular localization of DVDMS. Eca-109 cells were incubated with 5 lg mL
1 DVDMS for 3 h, and then coloaded with 2.5 nM Mito Tracker Green (MTG), a well-established fluorescent probe for mitochondria. After being washed twice with cold PBS, the cells were observed for the subcellular localization patterns of DVDMS under a laser scanning confocal microscopy (LSCM, Model TCS SP5; Leica, Germany). Phototoxicity of DVDMS and photofrin. Eca-109 cells (2 9 105 cells mL
1) were incubated with 5 lg mL
1 DVDMS or photofrin in 24-well culture plates. The cells were exposed to the light in different light dose (5.2, 10.4, 15.6 J cm
2). Then the cells were plated on 96-well culture plates (Corning Inc.). After 24 h, the cell viability was determined by adding 20 lL MTT solution (2.5 mg mL
1 in PBS) to each well and the mixture was incubated for 4 h at 37°C in a 5% CO2 incubator. After incubating, the mixture was removed and 150 lL pure DMSO was added per well. After shaking for 15 min at room temperature, the absorbance at 570 nm was recorded using a microplate reader (ELX800; Bio-Tek) against the reference value at 630 nm. The cell viability of treated cell samples was then obtained by comparing to the incubated nontreated control. For inhibitory experiments, 5 mM N-acetylcysteine (NAC), an ROS scavenger, was added to the medium for 1 h before loading DVDMS or photofrin. The inhibitor at the used concentration did not show any cell damage to cultured cells.

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Scanning electron microscopy (SEM) observation. After 24 h following PDT treatments, cells in control and PDT group were fixed with 2.5% glutaraldehyde in 0.01 M PBS (pH 7.2) for 24 h, and dehydrated by graded alcohol after being washed by PBS, then displaced and dried at the critical point. A thin layer of gold was evaporated onto the surface before observation using a scanning electron microscopy (S-3400N; Hitachi, Tokyo, Japan). Analysis of DNA fragmentation by flow cytometry. Krysko et al. described an easy and quantitative way to analyze DNA fragmentation based on flow fluorocytometric detection of DNA hypoploidy after adding PI to the dying cells and permeabilizing them by freeze-thawing. PI intercalates in the DNA and the size of DNA fragments appears as a hypoploid DNA histogram (42). To investigate the effect of DVDMSPDT on DNA damage of Eca-109 cells, we performed oligonucleosomal DNA fragmentation by flow cytometry. At 24 h posttreatment, the cells were stained with 5 lg mL
1 PI and freeze-thawed once by placing them briefly in liquid nitrogen. This procedure permeabilizes the cells and stains them with PI. Then the samples were analyzed by flow cytometry. Histograms were analyzed using FCS Express V3 software. Determination of intracellular ROS. Intracellular ROS production was studied by measuring the fluorescence intensity of dichlorofluorescein (DCF) as described in our previous papers (43). 2,7-DCF-diacetate (DCFH-DA), a nonfluorescent cell-permeant compound, is hydrolyzed by endogenous esterases within the cell and the de-esterified product can be converted into the fluorescent compound DCF upon oxidation by intracellular ROS. It has been reported the specificity of DCF is quite broad,

1 with a spectrum that includes H2O2, ˙OH, ˙O
2 , ONOO , OCl and O2 (44) and it has been used as a common probe for intracellular ROS detection in PDT studies (45–48). To estimate the intracellular ROS, we added 10 lM of DCFH-DA before PDT treatment, and at 2 h posttreatment, we harvested the cells, detected and analyzed DCF fluorescence by flow cytometry. For inhibitory experiments, cells were pretreated with 5 mM NAC, and then apply to the same protocol mentioned above to measure the intracellular ROS. Determination of mitochondrial membrane potential. To study the mitochondrial membrane potential (MMP) variation of Eca-109 cells after treatments, cells were stained with Rho123, which selectively enters mitochondria with an intact membrane potential and is retained in the mitochondria (49). Once the mitochondria membrane potential is lost, Rho123 is subsequently washed out of the cells. At 2 h after different treatments, cells were harvested and washed with PBS then incubated at 37°C with 1 lg mL
1 Rho123 for 20 min followed by washing with PBS. Then samples were immediately detected by flow cytometry. Statistical analysis. The SPSS 16.0 software (SPSS Inc., Chicago, IL) was used for the statistical analysis. Values are expressed as the mean  SD of three samples obtained from three independent experiments. Statistical comparisons were made using one-way analysis of variance (ANOVA). Intra or inter statistically significant differences were set at P < 0.05 or P < 0.01.

RESULTS Uptakes and intracellular localization of DVDMS The intracellular accumulation of DVDMS was evaluated by measuring the mean fluorescence intensity as determined by flow cytometry. As shown in Fig. 2, cellular uptake of DVDMS was relatively rapid, occurring within 2 h and reaching peak levels by 3 h. No further significant uptake was observed over the subsequent several hours. So in the following experiments, we chose 3 h as the drug-loading time. In addition, to assess the subcellular localization pattern of DVDMS in Eca-109 cells, we coloaded cells with mitochondrial specific dye MTG at 3 h after incubation with DVDMS. The fluorescence distributions of DVDMS and MTG were captured using laser scanning confocal microscopy. As shown in Fig. 3, the red fluorescence pattern of DVDMS corresponded well with that of MTG green fluorescence, indicating DVDMS mainly localized in the mitochondria of cells.

Figure 2. Cellular uptake of DVDMS in Eca-109 cells. Data are means  SD of three independent experiments. **P < 0.01 represents the DVDMS fluorescence intensity at 0.5 h versus 0 h. ##P < 0.01 represents the DVDMS fluorescence intensity at 1 h versus 0.5 h. MP < 0.05 represents the DVDMS fluorescence intensity at 2 h versus 1 h. ¤ P < 0.05 represents the DVDMS fluorescence intensity at 3 h versus 2 h.

Phototoxicity of DVDMS and photofrin on Eca-109 cells Data in Fig. 4 showed the cytotoxicity of Eca-109 cells after DVDMS-PDT (a) and photofrin-PDT (b). Obviously, coincubation of Eca-109 cells with DVDMS or photofrin without light exposure did not result in cytotoxicity under our experimental conditions (The cell viability was all above 97%). The light alone also did not effect the cell viability. Besides, the phototoxicity of DVDMS and photofrin were all enhanced by the increase of light dose. When the light dose was 10.4 J cm
2, the cell viability in DVDMS-PDT group was approximately 50% (P < 0.01 versus control and DVDMS alone). In addition, the cell viability in photofrin-PDT group was approximately 80%. (P < 0.01 versus control and P < 0.05 versus photofrin alone). Considering the statistical analysis of obtained data and the controllability of experimental system, 10.4 J cm
2 light dose was selected in the following experiments. These obtained results demonstrated that DVDMS could be effectively activated by light, and it had a much higher phototoxicity than photofrin, which had been approved by the FDA as a photosensitizer in PDT of cancer treatments. Scanning electron microscopy (SEM) observation The effect of DVDMS-PDT was morphologically observed under SEM (Fig. 5). In control group, cells appeared their normal polygon shape with numerous microvilli on their cell membrane surface. Whereas the cells treated with PDT, obviously cells shrunk and seriously decrease microvilli number were displayed. This exhibited that after PDT treatments, cells were seriously damaged. DVDMS-PDT-induced DNA damage As described in the methods, the DNA fragment was detected by flow cytometry. The obtained results (Fig. 6) showed that after

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Figure 3. Subcellular localization of DVDMS in Eca-109 cells.

Figure 4. (a) Phototoxicity of DVDMS on Eca-109 cells. (b) Phototoxicity of photofrin on Eca-109 cells. Data are means  SD of three independent experiments. *P < 0.05 and **P < 0.01 versus control. #P < 0.05 and ##P < 0.01 versus DVDMS or photofrin alone (light dose is 0 J cm
2).

24 h of incubation posttreatment, the DNA fragment of control is 2.33%, whereas those treated by DVDMS alone and light alone were 12.87% and 4.97%, and the DNA fragment increased to 58.37% post PDT treatment (P < 0.01 versus the other three groups). These results revealed that DVDMS-PDT markedly induced DNA damage of Eca-109 cells.

analyses results indicated that the generation of ROS in DVDMS-PDT group and photofrin-PDT group decreased after the cells pretreated with NAC. Furthermore, as shown in Fig. 7B, while pretreated by NAC, the viability of Eca-109 cells in DVDMS-PDT group and photofrin-PDT group all increased to above 80%. The results suggested ROS played an important role in PDT induced cytotoxicity on Eca-109 cells.

The involvement of intracellular ROS level in PDT Proposed mechanisms of PDT-induced cell death mainly focus on the generation of intracellular ROS (43). Therefore, we measured the intracellular ROS level using DCFH-DA to clarify whether the photodynamic effect of DVDMS was related to the alteration of ROS level in Eca-109 cells. According to the flow cytometry analyses (Fig. 7A), exposure of cells to DVDMS-PDT treatment significantly enhanced the intracellular ROS level (the mean fluorescence intensity of DCF in PDT group was much higher than control group and DVDMS group, P < 0.01 versus the other three groups). Compared with DVDMS-PDT, photofrin-PDT also generated a certain degree of ROS. The percentage of ROS in DVDMS-PDT group was 3.5 times higher than photofrin-PDT group (P < 0.01 between the two groups). To further investigate the role of ROS in PDT induced cell death in Eca109 cells, cells were pretreated with NAC. The flow cytometry

DVDMS-PDT-induced mitochondrial dysfunction Fig. 3 showed that DVDMS colocalized well with mitochondria, suggesting that mitochondria may be one of the dominating target of PDT. To detect the mitochondrial damage, we adopted Rho123 staining with flow cytometry to evaluate MMP changes induced by DVDMS-PDT. The mean fluorescence intensity of Rho123 directly proportional to MMP was used as an indicator. Results in Fig. 8 showed that compared with control, light alone could not cause obvious MMP change, DVDMS alone caused a certain degree of loss in MMP in which about 8.37% of cells showed low Rho123 fluorescence, whereas in PDT, the loss in MMP was further enhanced, and the percentage of cells with low Rho123 fluorescence increased to 16.43% (P < 0.01 versus the other three groups). The gained data indicated the occurrence of mitochondrial dysfunction after DVDMS-PDT treatments.

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Figure 5. Scanning electron microscopic images of Eca-109 cells at 24 h after different treatments. (a) the control group in 10009 magnification; (b) the DVDMS-PDT group in 10009 magnification; (c) the control group in 50009 magnification; (d) the DVDMS-PDT group in 50009 magnification.

Figure 6. DNA fragmentation of Eca-109 cells at 24 h after different treatments. (a) Control; (b) Light alone; (c) DVDMS alone; (d) DVDMS-PDT. Data shown are representative of three independent experiments. *P < 0.05 and **P < 0.01 versus control. ##P < 0.01 versus DVDMS or light alone.

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Figure 7. (A) Measurement of intracellular ROS in Eca-109 cells. (a) Control; (b) Light alone; (c) DVDMS alone; (d) DVDMS-PDT; (e) DVDMSPDT+NAC; (f) Photofrin alone; (g) Photofrin-PDT; (h) Photofrin-PDT+NAC. Data shown are representative of three independent experiments. *P < 0.05 and **P < 0.01 represents PDT versus control. ##P < 0.01 represents PDT versus DVDMS/photofrin or light alone. MMP < 0.01 represents PDT + NAC versus PDT. &&P < 0.01 represents photofrin-PDT group versus DVDMS-PDT group. (B) Effect of the ROS scavenger NAC on PDTinduced cytotoxicity in Eca-109 cells. Data are means  SD of three independent experiments. *P < 0.05 and **P < 0.01 compared with the same group without NAC.

DISCUSSION As a minimally invasive therapeutic approach, PDT has been widely used in the treatment of various tumors since the 1990s. Its efficacy as a curative and palliative therapeutic alternative is well documented (25,50,51). Undoubtedly, photosensitizer is a key component in the process of PDT development. However,

up to now, only photofrin has been widely approved and used by many countries in clinical cancer therapies. Obviously, appearance of new photosensitizers being developed will not only extend the number of choices for treating cancers already treated with photofrin, but extend the indications as well. According to previous reports, an ideal for PDT should meet several criteria: (1) chemical stability, (2) water-solubility, (3)

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Figure 8. Detection of mitochondrial membrane potential at 2 h after different treatments. (a) Control; (b) Light alone; (c) DVDMS alone; (d) DVDMS-PDT. Data shown are representative of three independent experiments. *P < 0.05 and **P < 0.01 versus control. ##P < 0.01 versus DVDMS or light alone.

high quantum yield of 1O2 generation,(4) no cytotoxicity in the dark, (5) tumor selectivity, (6) rapid accumulation in target tumor tissues, (7) rapid clearance from patients, (8) a high molar absorption coefficient in the long wavelength (600–800 nm) that can penetrate deeper tissues, etc (25,52,53). As we mentioned above, Professor Qicheng Fang had illustrated that DVDMS has an advantage of a 98.5% chemical purity, high solubility in water and short-time skin sensitivity. Besides, in our previous reports, DVDMS was found to shown higher fluorescence intensity and singlet oxygen production efficiency and could be selectively accumulated by tumor cells and tissues (39–41). In addition, the uptake and distribution of DVDMS at various time points in mice indicated that after DVDMS injection, it was widely distributed and had a long retention time in tumor tissues compared with normal tissues. Especially, 12 h and 24 h postinjection of DVDMS appeared to provide a higher therapeutic ratio (tumor versus skin or muscle). Therefore, PDT therapy between 12 and 24 h could maximize its therapeutic effect on tumors and minimize its side-effect on surrounding healthy tissues (37). These features laid a very good foundation for the application of DVDMS in PDT. However, how it does work has not been explored. Therefore, Eca-109 cells were selected as cell models to measure the phototoxicity of DVDMS and explored the possible mechanisms in this study. First, in PDT studies, the drug-loading time in cells is potentially critical for the therapeutic effect. In this study, Fig. 2 illustrated the intracellular cumulant of DVDMS reached a relatively

high level at 3 h, suggesting that DVDMS co-incubated with Eca-109 cells for 3 h, which may be the optimal time for light irradiation. Next, decreased cell viability was detected on Eca109 cells by DVDMS-PDT treatments. In addition, compared with photofrin which had been approved by the FDA as a photosensitizer in PDT of cancer treatments, DVDMS exhibited higher phototoxicity under the same experiment conditions (Fig. 4). Besides, the morphological changes of cells by SEM observation (Fig. 5) and DNA fragmentation by flow cytometry (Fig. 6) also showed that cells were seriously damaged and the DNA was also affected by DVDMS-PDT treatments. Hence, we demonstrated here that PDT with DVDMS had potent cytotoxicity on human esophageal cancer Eca-109 cells. Second, molecular pathways of cellular death are influenced by the intracellular distribution of the cytotoxic agent. Mitochondria are key organelles that regulate both cell survival and death. These organelles migrate through the cell, undergoing continuous fusion (elongation) and fission (fragmentation) processes (dynamics) to maintain proper function and meet cellular energy demands (energetics) (54,55). Confocal microscopy showed that DVDMS primarily accumulated in the mitochondria of Eca-109 cells (Fig. 3), indicating mitochondria was the main target for photochemical and mitochondria damage might be the major cause for DVDMS-induced cytotoxicity of Eca-109 cells. Energy demand during normal function is supplied by ATP, which is generated from the proton motive force (DlH), created by proton (DpH) and electrochemical gradients (DΨm) established by

Photochemistry and Photobiology, 2014, 90 electron movement through the respiratory chain (56–58). In PDT studies, increasing evidences has shown that changes of mitochondria membrane potential (MMP) are linked to phototoxicity (59–61). Subsequently, we monitored an initial MMP drop in Eca-109 cells after DVDMS-PDT (Fig. 8), indicating disaggregation of MMP and functional impairment of mitochondrial after PDT treatment. It is consistent with previous reports. In addition, investigations also show that PDT-induced cytotoxicity is closely related with the generation of ROS (43,62). In our study, we also found a rapid generation of intracellular ROS in PDT group, whereas there was no obvious occurrence in the control and DVDMS or photofrin alone groups. In addition, the generation of ROS in DVDMS-PDT group was much higher than photofrin-PDT group (Fig. 7A). It may be one of the reasons that DVDMS had a higher phototoxicity than photofrin. Moreover, coadministration with NAC, a special scavenger of ROS, the generation of ROS decreased and cell cytotoxicity effect of Eca-109 cells induced by PDT was inhibited (Fig. 7B). These results suggest an oxidative stress mechanism may be involved in response to PDT in Eca-109 cells. It appears that the principal mechanism of action of DVDMS activated by light or stems from the generation of ROS and loss in MMP. In conclusion, our results strongly support the notion that the photosensitizer, DVDMS-mediated PDT, may be a promising approach for the management of malignant tumors, and the phototoxicity is closely associated with ROS generation and MMP loss. However, the studies in vitro are somewhat limited. Especially, the PDT effect in various tumor-bearing models, pharmacokinetic study in various animals and safety evaluation are all key issues in their development process and clinical trials which must be strictly performed to carry out a series of rigorous assessments before it is used in the clinical cancer therapy. Therefore, further studies concerning the DVDMS evaluation and its PDT effects as well as detailed mechanisms need to be done in vivo. Acknowledgements—This work was supported by the project “DVDMS mediated sonodynamic therapy in anti-cancer research” with Qinglong Hi-tech Co., Ltd (Jiangxi, China). The authors thank Professor Qicheng Fang and Lecturer Haiyan Wu at the Chinese Academy of Medical Sciences for their contribution to this research.

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