J Cancer Res Clin Oncol DOI 10.1007/s00432-015-1918-1
ORIGINAL ARTICLE – CANCER RESEARCH
In vitro and in vivo antitumor activity of a novel porphyrin‑based photosensitizer for photodynamic therapy Jing‑Jing Chen · Ge Hong · Li‑Jing Gao · Tian‑Jun Liu · Wen‑Jun Cao
Received: 29 September 2014 / Accepted: 12 January 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose Photodynamic therapy (PDT) is a promising treatment in cancer therapy, based on the use of a photosensitizer activated by visible light in the presence of oxygen. Nowadays significant research efforts have been focused on finding a new photosensitizer. In the present paper, the antitumor effects of a novel porphyrin-based photosensitizer, {Carboxymethyl-[2-(carboxymethyl-{[4-(10,15,20-triphenylporphyrin-5-yl)-phenylcarbamoyl]-methyl}-amino)ethyl]-amino}-acetic acid (ATPP-EDTA) on two types of human malignant tumor cells in vitro and a gastric cancer model in nude mice, were evaluated. Methods The PDT efficacy with ATPP-EDTA in vitro was assessed by MTT assay. The intracellular accumulation was detected with fluorescence spectrometer, and the intracellular distribution was determined by laser scanning confocal microscopy. The mode of cell death was investigated by Hoechst 33342 staining and flow cytometer. BGC823derived xenograft tumor model was established to explore the in vivo antitumor effects of ATPP-EDTA.
Results ATPP-EDTA exhibited intense phototoxicity on both cell lines in vitro in concentration- and light dosedependent manners meanwhile imposing minimal dark cytotoxicity. The accumulation of ATPP-EDTA in two malignant cell lines was time-dependent and prior compared to normal cells. It was mainly localized at lysosomes, but induced cell death by apoptotic pathway. ATPP-EDTA significantly inhibited the growth of BGC823 tumors in nude mice (160 mW/cm2, 100 J/cm2). Conclusions Present studies suggest that ATPP-EDTA is an effective photosensitizer for PDT to tumors. It distributed in lysosomes and caused cell apoptosis. ATPP-EDTA, as a novel photosensitizer, has a great potential for human gastric cancer treatment in PDT and deserves further investigations. Keywords ATPP-EDTA · Photodynamic therapy · Intracellular localization · Apoptosis · Antitumor
Introduction
J.‑J. Chen · W.‑J. Cao Central Laboratory of Heping Hospital, Changzhi Medical College, Changzhi City 046000, Shanxi Province, People’s Republic of China G. Hong · T.‑J. Liu (*) Institute of Biomedical Engineering, Peking Union Medical College, Chinese Academy of Medical Sciences, Tianjin City 300192, People’s Republic of China e-mail: [email protected] L.‑J. Gao Department of Physiology of Changzhi Medical College, Changzhi City 046000, Shanxi Province, People’s Republic of China
Photodynamic therapy (PDT) is an effective treatment for malignant tumors imposing minimal damage to the surrounding healthy tissues (Celli et al. 2010). It typically involves the combination of a non-toxic photosensitizer with a visible light at an appropriate wavelength in the presence of oxygen, which could result in the generation of reactive oxygen species (ROS) and lead to malignant cells death via cellular damage, vasculature damage or recruiting members of the inflammatory and immune response system (Vrouenraets et al. 2003; Grimm et al. 2011; Moon et al. 2010). Compared to traditional chemotherapy, PDT has the advantage of being selective in destroying tumor tissues, since most types of tumors are especially active in
13
J Cancer Res Clin Oncol
Fig. 1 Chemical structure and spectrum properties of ATPP-EDTA in PBS. a Chemical structure of ATPP-EDTA in PBS. b Emission spectrum of ATPP-EDTA, which was excited at 422 nm, and its peaks were at 658 and 720 nm. c–d UV–Vis absorption spectrum of ATPP-EDTA in PBS. Its maximum absorbance is at 422 nm, and at 518, 554, 592 and 648 nm, also it has absorption
both the uptake and accumulation of photosensitizers than normal tissues, which makes tumors especially vulnerable to PDT (Selbo et al. 2002). The selectivity also could be achieved by illumination spatially directed to the tumor tissues (Konan et al. 2002; Pandey et al. 2006). Because of the high reactivity and the limited scope of diffusion of ROS, subcellular distribution of a photosensitizer often determines the primary sites and the type of photodamage (Kessel et al. 2003; Oleinick et al. 2002). Photosensitizers can localize within many different cellular organelles such as the mitochondria, lysosomes, plasma membranes, endoplasmic reticulum and Golgi apparatus (Pazos and Nader 2007; Morgan and Oseroff 2001). Mitochondrion is the well-known apoptotic initiator and often induces the intrinsic pathway of apoptosis (Liu et al. 2013). These mitochondrion-targeted photosensitizers could initiate cell apoptosis by mitochondrial damage after light illumination (Runnels et al. 1999). With PS localized in the plasma membrane, the photosensitization process can rapidly lead to necrotic cell death due to the loss of plasma membrane integrity and rapid depletion of intracellular ATP (Kessel and Poretz 2000). Recently, many photosensitizers distributed in lysosomes have been used in photodynamic therapy such as N-aspartyl chlorin e6 (Npe6; Almeida et al. 2004; Buytaert et al. 2007; Liu et al. 2011). The combination of light and such photosensitizers causes lysosomal photodamage and release of proteolytic enzyme, subsequently inducing cell necrosis (Kessel and Sun 1999). However, it has been suggested that photosensitizers that
13
accumulate in lysosomes might also induce apoptosis (Kessel et al. 2000). In fact, lysosome plays a far more sophisticated role in cell death caused by PDT than was previously thought. Light, photosensitizer and oxygen are the three basic elements for PDT, among which photosensitizer is the most important factor. {Carboxymethyl-[2-(carboxymethyl-{[4(10,15,20-triphenylporphyrin-5-yl)-phenylcarbamoyl]methyl}-amino)-ethyl]-amino}-acetic acid (ATPP-EDTA; Fig. 1a) is a pure and novel porphyrin-based photosensitizer synthesized by our laboratory according to our patent specifications. The photodynamic effects and related mechanisms about this compound on malignant tumor cells were unclear. In the present study, the photophysical and photochemical properties of ATPP-EDTA were showed and the phototoxicity was evaluated in vitro and in vivo. To further elucidate the mechanisms of action, the uptake, intracellular localization of ATPP-EDTA and the mode of cell death by ATPP-EDTA-PDT are investigated as well.
Materials and methods Preparation of ATPP‑EDTA Synthesis of 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (ATPP) was realized according to the procedure reported by Luguya (Luguya et al. 2004). ATPP-EDTA was synthesized according to our patent specification (No.
J Cancer Res Clin Oncol
ZL201010184331.7) (Liu et al. 2010). First, we dissolved 51.2 mg of EDTA dianhydride and 100 mg ATPP in 20 Ml of dry dimethyl formamide (DMF), added 50 μL of triethylamine and then stirred the solution in nitrogen atmosphere at room temperature for 12 h. We next added 40 mL of cold deionized water to the mother liquor, and then, crude product was obtained by centrifugation separating the precipitation. The solid was subjected to column chromatography using silica gel (200 mesh) and eluted with a mixture of chloroform and methanol. The final pure ATPP-EDTA product was a dark marron powder. Yield 0.123 g (85 %), mp > 300 °C (decomposed). IR (KBr): ν, cm−1 3,313 (OH), 1,721 (C=O), 1,680 (HNC=O). HRMS (MALDI): m/z 904.3458 (calcd. for [M+H] + 904.3453). IR spectra were recorded on a Thermo fisher Nicolet infrared spectrometer in KBr pellets (Model iS10, USA). High-resolution mass spectra were recorded on a Bruker MALDI-TOF mass spectrometer (Model Autoflex III TOF/TOF200, Germany). Chemicals All the chemicals and reagents used in the synthesis of ATPP-EDTA were obtained from Sigma-Aldrich. They were of analytical grade and used without any purification. Absorption and emission spectra UV–Vis absorption spectrum with ATPP-EDTA was recorded on an ultraviolet visible spectrophotometer (Thermo 3001, Finland). Fluorescence spectra were measured on a Fluorescence Spectrometer (F7000, Japan). Slits were kept narrow to 1 nm in excitation and 1 or 2 nm in emission. ATPP-EDTA was dissolved in phosphate-buffered saline (PBS; Solarbio, China) to get a 3.12 μM solution. Cells Human liver carcinoma cell line (HepG2 cell), human gastric carcinoma cell line (BGC823 cell) and human normal liver cell line (LO2 cell) were obtained from the Chinese Academy of Sciences and cultured in normal RPMI-1640 culture medium (Solarbio, China) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA). Cells were maintained in 5 % CO2 and 21 % O2 at 37 °C. Accumulation of ATPP‑EDTA in cells HepG2, BGC823 and LO2 cells (1 × 104 cells/well) were seeded into 96-well cell culture plates for 24 h, followed by incubation with ATPP-EDTA at a concentration of 3.12 μM for various time intervals, from 4 up to 24 h. At the end of each incubation, the cells were washed with
PBS and solubilized in 100 μL of 0.25 % Triton X-100 (Amresco 0694, USA). The retention of cell-associated ATPP-EDTA was measured by fluorescence using excitation/emission wavelength of 422/658 nm, respectively. The amount of ATPP-EDTA uptaken by cells was expressed as μm/104 cells and calculated according to the calibration curve (fluorescence vs. concentration). MTT cell viability assay Cell viability was assessed by using the 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Beyotime, China) colorimetric assay. HepG2 and BGC823 cells were incubated with ATPP-EDTA at concentrations ranging from 0.78 to 12.5 μM for 24 h. The supernatant drug solution was removed and fresh 1640 culture medium was added. The cells were then irradiated by a laser (Intense Model 7404, USA) at 650 nm with various light doses of 3, 6, 12 J/cm2. After a 3-h incubation post-illumination, MTT solution (5 mg/mL) in 20 μL of PBS was added into each well for another 1 h. The reaction was finally stopped by the addition of 180 μL DMSO (Solarbio, China), and the degree of color development was measured by a UV–Visible spectrophotometer (Thermo 3001, Finland) at a wavelength of 490 nm. Cells exposed to ATPP-EDTA alone but not to light illumination (dark cytotoxicity) were used as a control. Cell survival (%) = (mean OD value of treated cells/mean OD value of control cells) × 100 %. Each individual phototoxic experiment was repeated for four times. Intracellular localization Cells cultured in each 35-mm Petri dish were incubated with ATPP-EDTA at a concentration of 3.12 μM for 24 h in the dark. After washing with PBS, the cells were stained with fluorescent dye (150 nM MitoTracker Green FM (Invitrogen, USA) staining mitochondrion for 30 min or 1.5 μM LysoSensor™ Green DND-189 (Invitrogen, USA) staining lysosome for 1 h in the dark at room temperature). After washing with PBS once more, the cells were examined by fluorescence with a confocal laser scanning microscopy (CLSM; Leica Heidelberg GmbH, Germany). The red fluorescence from ATPP-EDTA was excited at wavelength 405 nm, and its emission wavelength was monitored at wavelength 660 nm. The green fluorescence from MitoTracker Green FM or LysoSensor™ Green DND-189 was excited at wavelength 488 nm, and its emission was detected at 520 nm. The red fluorescence and green fluorescence were combined as the overlay fluorescence. In CLSM imaging of the two different types of tumor cells, all control parameters were kept constant throughout to ensure reliable comparison.
13
J Cancer Res Clin Oncol
Apoptotic detection BGC823 cells were loaded into 6-well plates overnight and then incubated with ATPP-EDTA at a concentration of 6.25 μM for 24 h. After that, the drug solution was replaced with a fresh culture medium, and the cells were irradiated by a laser (Intense Model 7404, USA) at 650 nm with a fluence of 6 J/cm2. After 3 h, the irradiated cells were collected, rinsed with PBS and stained with 200 μg/mL Annexin V-FITC (Beyotime, China) for 10 min and subsequently with 30 μg/mL PI (Beyotime, China) for immediate at room temperature in the dark. Immediately, the fluorescence was analyzed in 10,000 cells per sample by using a FAC-Scan flow cytometer (Beckman, USA). The results were expressed as the percentage of cells exhibiting apoptosis relative to the total number of cells analyzed. The same treated HepG2 and BGC823 cells with ATPP-EDTA incubation and light illumination were stained with Hoechst 33342 (Beyotime, China) for cell nucleus for 30 min at room temperature in the dark, then washed twice with PBS and photographed under a fluorescent microscope (Leica-DMIRE2, Germany) to detect the difference in chromatin condensation and fragmentation. The blue fluorescence from Hoechst 33342 was excited at 350 nm, and its emission was detected at 461 nm. Animal models Six-week-old female Balb C athymic nu/nu mice (Beijing HFK Bioscience, China) were fed under standard conditions and prepared for in vivo experiment. 1 × 106 BGC823 cells in 100 μL PBS were subcutaneously injected into the right posterior limbs of mice under anesthesia. The tumor was allowed to grow to an approximate volume of 100 mm3. All animal experiments were approved by the review committee for the use of animal subjects of Changzhi Medical College. In vivo PDT Experimental mice were randomly divided into four groups (six mice per group): control group (physiological saline, only once) and three doses of ATPP-EDTA group (1, 5, 10 mg/kg, only once). Physiological saline or ATPP-EDTA was administered intravenously via the caudal vein. PDT was performed at 24 h following ATPP-EDTA injection by a laser (Intense Model 7404, USA) at 650 nm with a light dose of 100 J/cm2 (160 mW/cm2) under anesthesia with thiopental (0.5 mg/kg). Tumor inhibition rate (%) = (WC– WD)/WC, where WC = tumor weight of control group, WD = tumor weight of drug-PDT group.
13
Fig. 2 Accumulation of ATPP-EDTA. The data shown are the mean ± SD from three independent experiments
Results UV–Vis absorption and emission spectrum Fluorescence spectra were measured using spectrofluorimeter as described in experimental section. ATPP-EDTA can be excited at 422 nm, and its emission was detected at wavelengths of 658 and 720 nm (Fig. 1b). The absorption peak of ATPP-EDTA in PBS is at 422 nm, lower peak is at 518 nm, and it also has absorption at 554, 592 and 648 nm (Fig. 1c, d). Since the absorption is the strongest at 422 nm, 405 nm nearest which could be chosen as the excitation laser channel during CLSM. Meanwhile, considering the penetration depth of light in PDT proportional to the wavelength, a long wavelength near the red region of the spectrum should be used for laser illumination during the treatment of PDT, and 650 nm is the suitable wavelength for the MTT and flow cytometry assays. Accumulation of ATPP‑EDTA in cells Cells were exposed to ATPP-EDTA for various time intervals, and intracellular drug accumulation was detected at the end of each incubation period. Figure 2 shows that ATPP-EDTA could effectively accumulate in HepG2 and BGC823 cells, and it was time dependent. In the first 12 h, the intracellular ATPP-EDTA accumulation increased rapidly in both cell lines, after which it almost reached a plateau even at 24 h. The time of 24 h could be used as the incubation time of ATPP-EDTA in the following experiments. Under similar conditions, ATPP-EDTA accumulated in HepG2 cells to a slightly higher extent than in BGC823 cells. Figure 2 also demonstrates that malignant cells had a preferential ATPP-EDTA uptake in comparison with LO2
J Cancer Res Clin Oncol
Fig. 3 Concentration–effect curves for ATPP-EDTA in HepG2 (a) and BGC823 cells (b) with different light dose. HepG2 and BGC823 cells were exposed to concentrations from 0 to 12.5 μM for 24 h; then, cells were irradiated at a fluence rate of total light doses rang-
ing from 3 to 12 J/cm2. Cell cytotoxicity was determined 3 h after the end of irradiation by MTT test. The data shown are the mean ± SD of four independent experiments
Table 1 IC50 values of ATPP-EDTA in HepG2 and BGC823 cells treated for 24 h and then exposed to increasing doses of red light (650 nm)
alone, except for those incubated with the highest concentration (12.5 μM) showing slight growth inhibition.
Cells
Intracellular localization
IC50 (μM) 2
2
2
3 J/cm
6 J/cm
12 J/cm
HepG2
4.02 ± 0.29*
1.49 ± 0.16*
1.13 ± 0.12*
BGC823
7.70 ± 1.43
2.82 ± 0.38*
1.26 ± 0.09*
Results represent mean ± SD of three independent experiments * P
ORIGINAL ARTICLE – CANCER RESEARCH
In vitro and in vivo antitumor activity of a novel porphyrin‑based photosensitizer for photodynamic therapy Jing‑Jing Chen · Ge Hong · Li‑Jing Gao · Tian‑Jun Liu · Wen‑Jun Cao
Received: 29 September 2014 / Accepted: 12 January 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose Photodynamic therapy (PDT) is a promising treatment in cancer therapy, based on the use of a photosensitizer activated by visible light in the presence of oxygen. Nowadays significant research efforts have been focused on finding a new photosensitizer. In the present paper, the antitumor effects of a novel porphyrin-based photosensitizer, {Carboxymethyl-[2-(carboxymethyl-{[4-(10,15,20-triphenylporphyrin-5-yl)-phenylcarbamoyl]-methyl}-amino)ethyl]-amino}-acetic acid (ATPP-EDTA) on two types of human malignant tumor cells in vitro and a gastric cancer model in nude mice, were evaluated. Methods The PDT efficacy with ATPP-EDTA in vitro was assessed by MTT assay. The intracellular accumulation was detected with fluorescence spectrometer, and the intracellular distribution was determined by laser scanning confocal microscopy. The mode of cell death was investigated by Hoechst 33342 staining and flow cytometer. BGC823derived xenograft tumor model was established to explore the in vivo antitumor effects of ATPP-EDTA.
Results ATPP-EDTA exhibited intense phototoxicity on both cell lines in vitro in concentration- and light dosedependent manners meanwhile imposing minimal dark cytotoxicity. The accumulation of ATPP-EDTA in two malignant cell lines was time-dependent and prior compared to normal cells. It was mainly localized at lysosomes, but induced cell death by apoptotic pathway. ATPP-EDTA significantly inhibited the growth of BGC823 tumors in nude mice (160 mW/cm2, 100 J/cm2). Conclusions Present studies suggest that ATPP-EDTA is an effective photosensitizer for PDT to tumors. It distributed in lysosomes and caused cell apoptosis. ATPP-EDTA, as a novel photosensitizer, has a great potential for human gastric cancer treatment in PDT and deserves further investigations. Keywords ATPP-EDTA · Photodynamic therapy · Intracellular localization · Apoptosis · Antitumor
Introduction
J.‑J. Chen · W.‑J. Cao Central Laboratory of Heping Hospital, Changzhi Medical College, Changzhi City 046000, Shanxi Province, People’s Republic of China G. Hong · T.‑J. Liu (*) Institute of Biomedical Engineering, Peking Union Medical College, Chinese Academy of Medical Sciences, Tianjin City 300192, People’s Republic of China e-mail: [email protected] L.‑J. Gao Department of Physiology of Changzhi Medical College, Changzhi City 046000, Shanxi Province, People’s Republic of China
Photodynamic therapy (PDT) is an effective treatment for malignant tumors imposing minimal damage to the surrounding healthy tissues (Celli et al. 2010). It typically involves the combination of a non-toxic photosensitizer with a visible light at an appropriate wavelength in the presence of oxygen, which could result in the generation of reactive oxygen species (ROS) and lead to malignant cells death via cellular damage, vasculature damage or recruiting members of the inflammatory and immune response system (Vrouenraets et al. 2003; Grimm et al. 2011; Moon et al. 2010). Compared to traditional chemotherapy, PDT has the advantage of being selective in destroying tumor tissues, since most types of tumors are especially active in
13
J Cancer Res Clin Oncol
Fig. 1 Chemical structure and spectrum properties of ATPP-EDTA in PBS. a Chemical structure of ATPP-EDTA in PBS. b Emission spectrum of ATPP-EDTA, which was excited at 422 nm, and its peaks were at 658 and 720 nm. c–d UV–Vis absorption spectrum of ATPP-EDTA in PBS. Its maximum absorbance is at 422 nm, and at 518, 554, 592 and 648 nm, also it has absorption
both the uptake and accumulation of photosensitizers than normal tissues, which makes tumors especially vulnerable to PDT (Selbo et al. 2002). The selectivity also could be achieved by illumination spatially directed to the tumor tissues (Konan et al. 2002; Pandey et al. 2006). Because of the high reactivity and the limited scope of diffusion of ROS, subcellular distribution of a photosensitizer often determines the primary sites and the type of photodamage (Kessel et al. 2003; Oleinick et al. 2002). Photosensitizers can localize within many different cellular organelles such as the mitochondria, lysosomes, plasma membranes, endoplasmic reticulum and Golgi apparatus (Pazos and Nader 2007; Morgan and Oseroff 2001). Mitochondrion is the well-known apoptotic initiator and often induces the intrinsic pathway of apoptosis (Liu et al. 2013). These mitochondrion-targeted photosensitizers could initiate cell apoptosis by mitochondrial damage after light illumination (Runnels et al. 1999). With PS localized in the plasma membrane, the photosensitization process can rapidly lead to necrotic cell death due to the loss of plasma membrane integrity and rapid depletion of intracellular ATP (Kessel and Poretz 2000). Recently, many photosensitizers distributed in lysosomes have been used in photodynamic therapy such as N-aspartyl chlorin e6 (Npe6; Almeida et al. 2004; Buytaert et al. 2007; Liu et al. 2011). The combination of light and such photosensitizers causes lysosomal photodamage and release of proteolytic enzyme, subsequently inducing cell necrosis (Kessel and Sun 1999). However, it has been suggested that photosensitizers that
13
accumulate in lysosomes might also induce apoptosis (Kessel et al. 2000). In fact, lysosome plays a far more sophisticated role in cell death caused by PDT than was previously thought. Light, photosensitizer and oxygen are the three basic elements for PDT, among which photosensitizer is the most important factor. {Carboxymethyl-[2-(carboxymethyl-{[4(10,15,20-triphenylporphyrin-5-yl)-phenylcarbamoyl]methyl}-amino)-ethyl]-amino}-acetic acid (ATPP-EDTA; Fig. 1a) is a pure and novel porphyrin-based photosensitizer synthesized by our laboratory according to our patent specifications. The photodynamic effects and related mechanisms about this compound on malignant tumor cells were unclear. In the present study, the photophysical and photochemical properties of ATPP-EDTA were showed and the phototoxicity was evaluated in vitro and in vivo. To further elucidate the mechanisms of action, the uptake, intracellular localization of ATPP-EDTA and the mode of cell death by ATPP-EDTA-PDT are investigated as well.
Materials and methods Preparation of ATPP‑EDTA Synthesis of 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (ATPP) was realized according to the procedure reported by Luguya (Luguya et al. 2004). ATPP-EDTA was synthesized according to our patent specification (No.
J Cancer Res Clin Oncol
ZL201010184331.7) (Liu et al. 2010). First, we dissolved 51.2 mg of EDTA dianhydride and 100 mg ATPP in 20 Ml of dry dimethyl formamide (DMF), added 50 μL of triethylamine and then stirred the solution in nitrogen atmosphere at room temperature for 12 h. We next added 40 mL of cold deionized water to the mother liquor, and then, crude product was obtained by centrifugation separating the precipitation. The solid was subjected to column chromatography using silica gel (200 mesh) and eluted with a mixture of chloroform and methanol. The final pure ATPP-EDTA product was a dark marron powder. Yield 0.123 g (85 %), mp > 300 °C (decomposed). IR (KBr): ν, cm−1 3,313 (OH), 1,721 (C=O), 1,680 (HNC=O). HRMS (MALDI): m/z 904.3458 (calcd. for [M+H] + 904.3453). IR spectra were recorded on a Thermo fisher Nicolet infrared spectrometer in KBr pellets (Model iS10, USA). High-resolution mass spectra were recorded on a Bruker MALDI-TOF mass spectrometer (Model Autoflex III TOF/TOF200, Germany). Chemicals All the chemicals and reagents used in the synthesis of ATPP-EDTA were obtained from Sigma-Aldrich. They were of analytical grade and used without any purification. Absorption and emission spectra UV–Vis absorption spectrum with ATPP-EDTA was recorded on an ultraviolet visible spectrophotometer (Thermo 3001, Finland). Fluorescence spectra were measured on a Fluorescence Spectrometer (F7000, Japan). Slits were kept narrow to 1 nm in excitation and 1 or 2 nm in emission. ATPP-EDTA was dissolved in phosphate-buffered saline (PBS; Solarbio, China) to get a 3.12 μM solution. Cells Human liver carcinoma cell line (HepG2 cell), human gastric carcinoma cell line (BGC823 cell) and human normal liver cell line (LO2 cell) were obtained from the Chinese Academy of Sciences and cultured in normal RPMI-1640 culture medium (Solarbio, China) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA). Cells were maintained in 5 % CO2 and 21 % O2 at 37 °C. Accumulation of ATPP‑EDTA in cells HepG2, BGC823 and LO2 cells (1 × 104 cells/well) were seeded into 96-well cell culture plates for 24 h, followed by incubation with ATPP-EDTA at a concentration of 3.12 μM for various time intervals, from 4 up to 24 h. At the end of each incubation, the cells were washed with
PBS and solubilized in 100 μL of 0.25 % Triton X-100 (Amresco 0694, USA). The retention of cell-associated ATPP-EDTA was measured by fluorescence using excitation/emission wavelength of 422/658 nm, respectively. The amount of ATPP-EDTA uptaken by cells was expressed as μm/104 cells and calculated according to the calibration curve (fluorescence vs. concentration). MTT cell viability assay Cell viability was assessed by using the 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Beyotime, China) colorimetric assay. HepG2 and BGC823 cells were incubated with ATPP-EDTA at concentrations ranging from 0.78 to 12.5 μM for 24 h. The supernatant drug solution was removed and fresh 1640 culture medium was added. The cells were then irradiated by a laser (Intense Model 7404, USA) at 650 nm with various light doses of 3, 6, 12 J/cm2. After a 3-h incubation post-illumination, MTT solution (5 mg/mL) in 20 μL of PBS was added into each well for another 1 h. The reaction was finally stopped by the addition of 180 μL DMSO (Solarbio, China), and the degree of color development was measured by a UV–Visible spectrophotometer (Thermo 3001, Finland) at a wavelength of 490 nm. Cells exposed to ATPP-EDTA alone but not to light illumination (dark cytotoxicity) were used as a control. Cell survival (%) = (mean OD value of treated cells/mean OD value of control cells) × 100 %. Each individual phototoxic experiment was repeated for four times. Intracellular localization Cells cultured in each 35-mm Petri dish were incubated with ATPP-EDTA at a concentration of 3.12 μM for 24 h in the dark. After washing with PBS, the cells were stained with fluorescent dye (150 nM MitoTracker Green FM (Invitrogen, USA) staining mitochondrion for 30 min or 1.5 μM LysoSensor™ Green DND-189 (Invitrogen, USA) staining lysosome for 1 h in the dark at room temperature). After washing with PBS once more, the cells were examined by fluorescence with a confocal laser scanning microscopy (CLSM; Leica Heidelberg GmbH, Germany). The red fluorescence from ATPP-EDTA was excited at wavelength 405 nm, and its emission wavelength was monitored at wavelength 660 nm. The green fluorescence from MitoTracker Green FM or LysoSensor™ Green DND-189 was excited at wavelength 488 nm, and its emission was detected at 520 nm. The red fluorescence and green fluorescence were combined as the overlay fluorescence. In CLSM imaging of the two different types of tumor cells, all control parameters were kept constant throughout to ensure reliable comparison.
13
J Cancer Res Clin Oncol
Apoptotic detection BGC823 cells were loaded into 6-well plates overnight and then incubated with ATPP-EDTA at a concentration of 6.25 μM for 24 h. After that, the drug solution was replaced with a fresh culture medium, and the cells were irradiated by a laser (Intense Model 7404, USA) at 650 nm with a fluence of 6 J/cm2. After 3 h, the irradiated cells were collected, rinsed with PBS and stained with 200 μg/mL Annexin V-FITC (Beyotime, China) for 10 min and subsequently with 30 μg/mL PI (Beyotime, China) for immediate at room temperature in the dark. Immediately, the fluorescence was analyzed in 10,000 cells per sample by using a FAC-Scan flow cytometer (Beckman, USA). The results were expressed as the percentage of cells exhibiting apoptosis relative to the total number of cells analyzed. The same treated HepG2 and BGC823 cells with ATPP-EDTA incubation and light illumination were stained with Hoechst 33342 (Beyotime, China) for cell nucleus for 30 min at room temperature in the dark, then washed twice with PBS and photographed under a fluorescent microscope (Leica-DMIRE2, Germany) to detect the difference in chromatin condensation and fragmentation. The blue fluorescence from Hoechst 33342 was excited at 350 nm, and its emission was detected at 461 nm. Animal models Six-week-old female Balb C athymic nu/nu mice (Beijing HFK Bioscience, China) were fed under standard conditions and prepared for in vivo experiment. 1 × 106 BGC823 cells in 100 μL PBS were subcutaneously injected into the right posterior limbs of mice under anesthesia. The tumor was allowed to grow to an approximate volume of 100 mm3. All animal experiments were approved by the review committee for the use of animal subjects of Changzhi Medical College. In vivo PDT Experimental mice were randomly divided into four groups (six mice per group): control group (physiological saline, only once) and three doses of ATPP-EDTA group (1, 5, 10 mg/kg, only once). Physiological saline or ATPP-EDTA was administered intravenously via the caudal vein. PDT was performed at 24 h following ATPP-EDTA injection by a laser (Intense Model 7404, USA) at 650 nm with a light dose of 100 J/cm2 (160 mW/cm2) under anesthesia with thiopental (0.5 mg/kg). Tumor inhibition rate (%) = (WC– WD)/WC, where WC = tumor weight of control group, WD = tumor weight of drug-PDT group.
13
Fig. 2 Accumulation of ATPP-EDTA. The data shown are the mean ± SD from three independent experiments
Results UV–Vis absorption and emission spectrum Fluorescence spectra were measured using spectrofluorimeter as described in experimental section. ATPP-EDTA can be excited at 422 nm, and its emission was detected at wavelengths of 658 and 720 nm (Fig. 1b). The absorption peak of ATPP-EDTA in PBS is at 422 nm, lower peak is at 518 nm, and it also has absorption at 554, 592 and 648 nm (Fig. 1c, d). Since the absorption is the strongest at 422 nm, 405 nm nearest which could be chosen as the excitation laser channel during CLSM. Meanwhile, considering the penetration depth of light in PDT proportional to the wavelength, a long wavelength near the red region of the spectrum should be used for laser illumination during the treatment of PDT, and 650 nm is the suitable wavelength for the MTT and flow cytometry assays. Accumulation of ATPP‑EDTA in cells Cells were exposed to ATPP-EDTA for various time intervals, and intracellular drug accumulation was detected at the end of each incubation period. Figure 2 shows that ATPP-EDTA could effectively accumulate in HepG2 and BGC823 cells, and it was time dependent. In the first 12 h, the intracellular ATPP-EDTA accumulation increased rapidly in both cell lines, after which it almost reached a plateau even at 24 h. The time of 24 h could be used as the incubation time of ATPP-EDTA in the following experiments. Under similar conditions, ATPP-EDTA accumulated in HepG2 cells to a slightly higher extent than in BGC823 cells. Figure 2 also demonstrates that malignant cells had a preferential ATPP-EDTA uptake in comparison with LO2
J Cancer Res Clin Oncol
Fig. 3 Concentration–effect curves for ATPP-EDTA in HepG2 (a) and BGC823 cells (b) with different light dose. HepG2 and BGC823 cells were exposed to concentrations from 0 to 12.5 μM for 24 h; then, cells were irradiated at a fluence rate of total light doses rang-
ing from 3 to 12 J/cm2. Cell cytotoxicity was determined 3 h after the end of irradiation by MTT test. The data shown are the mean ± SD of four independent experiments
Table 1 IC50 values of ATPP-EDTA in HepG2 and BGC823 cells treated for 24 h and then exposed to increasing doses of red light (650 nm)
alone, except for those incubated with the highest concentration (12.5 μM) showing slight growth inhibition.
Cells
Intracellular localization
IC50 (μM) 2
2
2
3 J/cm
6 J/cm
12 J/cm
HepG2
4.02 ± 0.29*
1.49 ± 0.16*
1.13 ± 0.12*
BGC823
7.70 ± 1.43
2.82 ± 0.38*
1.26 ± 0.09*
Results represent mean ± SD of three independent experiments * P
