Hematoporphyrin monomethyl ether is a promising porphyrin-related photosensitize for photodynamic therapy. There still remains unknown changes regarding the mitochondrial in canine breast cancer cells treated with HMME-PDT. The aim of this study is to investigate the effect of HMME-PDT on structure and dysfunction of mitochondrial in cancer cells. The experimental approach included an initial study on the uptake of HMME using microscopic observation of the HMME-treated cells, optimization of the PDT-induced cell death by the MTT assay. These cells were then treated with HMME and a He-Ne laser at the wavelength of 632.8 nm following our optimized condition. examination of mitochondrial changes by observing the stained cells under light microscope, mitochjondrial membrane potential flow cytometry, measuring the Ca2+, SOD/GSH activity, ATPase and MDA contents for the mitochondria functions. The kinetics of HMME uptake in CHMm cells was determined and its cytocolic instead of nuclear distribution was demonstrated. The dose of 16mM HMME-PDT combined with 2.8 Jâˆ™cm-2 laser irradiation was had the maximal impact on cell viability. This treatment resulted in structural changes in mitochondria that were accompanied with the loss of mitochjondrial membrane potential. As a result, HMME-PDT increased mitochondrial ROS, inhibited the enzymatic activities of mitochondrial SOD and GSH-Px, abolished mitochondrial ability in the uptake and release of calcium, and decreased mitochondrial ATPase activity. The combination of these abnormalities led to accumulation of ROS in mitochondrial to high levels, which in turn contributed to HMME-PDT-induced damages of mitochondrial structure and mitochondrial dysfunction.
Hematoporphyrin monomethyl ether, CHMm cells, Mitochondrial dysfunction, mitochondrial structure, PDT
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Mammary gland tumors are amongst the most common neoplasms in humans, likewise in dogs. The underlying occurrence mechanism of the tumor has been frond to be similar in the two mamals . Canine and human mammary tumors share several important epidemiological, morphological, clinicopathological and biochemical features . The spontaneous canine mammary tumor as an increasingly powerful model has been used to study on human breast cancer . Therefore, it is of great important to investigate and evaluate possible treatment for dog mammary tumor. Additionally, a better and less painfull treatment for dog mammary tumor should be concerned for animal welfare since increasing incidence of the diseases. A recent publication in Norway reported on a high frequency (53.3%) of breast carcinomas in 14,401 investigated dogs .
Hematoporphyrin monomethyl ether (HMME) is a promising second-generation porphyrin-related photosensitizer for PDT. Experimental studies and clinical trials have demonstrated that HMME can be selectively taken by tumor tissues. It has a strong photodynamic effect, with lower toxicity, shorter-term skin photosensitizations and less costly . To date, HMME has been tested for clinical trial as a treatment for skin diseases. It has been evaluated in the clinical trials of Port wine stains (PWS) in china [6-8]. It has also been reported to be an effective and safe modality for treating PWS . In addition, Recently, HMME, as a promising photosensitizer, was utilized for antitumor PDT in vitro and in vivo studies. It induces cervix cancer cell death through both necrosis and apoptosis . HMME-based PDT is proved to be an effective way of treatment for ovarian cancer, cutaneous malignancy, human glioblastoma cells [9, 11, 12]. Although preclinical and clinical evaluations suggest that HMME is a promising PDT photosensitizer for human disease treatment, there is comparatively little published thus far on investigation of animal tumor thus far.
Substantial amount of evidence suggests that mitochondria play a major role in HMME-PDT inducing apoptosis in cancer cells [10, 11, 13, 14]. Mechanisms of cell death response to photodynamic therapy are dependent on different dose and cell types [15, 16]. While HMME-PDT can induce apoptosis of canine breast cancer cells , the effect of the treatment on mitochondrial remains unknown Therefore, we optimized the treatment condition of HMME-PDT, and investigated the effects of the treatment on the function and structure of mitochondrial.
Materials and methods
Reagents and cell culture
Hematoporphyrin monomethyl ether was provided by RedGreen Photosensitizer Co., Ltd. (Shanghai, China). A stock solution was made in ethanol at a concentration of 10 mg/ml and kept in the dark at −20 °C. The He-Ne laser, equipped with a 632.8 nm wavelength and an output power â©¾30 mW, was purchased from Kinglaser Technology (Jilin, China). CHMm cells were isolated from metastatic lesions of a 12 year-old female dog. Cells have been maintained for more than 60 generations without noticeable changes in their morphologies and proliferation characteristics . CHMm cells were maintained in DMEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum at 37â„ƒ under a humidified atmosphere containing 5% carbon dioxide.
HMME uptake and its cellular distribution in CHMm cells
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CHMm cells were seeded in dishes (35 mm diameter) at 1-105 cells/dish and cultured for 24 hours in a complete medium. When cells were at the phase of exponential growth, the medium was replaced with serum free DMEM containing 196mM HMME. Cells were cultured for 10, 15, 30, 45, 60, 90, 120, 240 min in the dark in a tissue-culture incubator at 37â„ƒ and 5% CO2. Cells were then rinsed twice with PBS and examined under a fluorescence microscope. The fluorescence emission of HMME was simultaneously collected from BA-515nm filter with a DM-505nm spectroscope under excitation at 465-495 nm (BP 465-495). Photobleaching of HMME was collected from BA-420nm filter under fluorescence microscope with a DM-400 spectroscope. Cells incubated for 120 min with 196mM HMME, and then irradiated for 0, 5, or 10 min at 330-380nm (BP 330-380) under 100W high-voltage mercury lamp, and the power was 10mW/cm2.
Optimization of HMME-PDT treatment
Cells were seeded in 96-well plates at a density of 1-104 cells/well and cultured for 24 h in a complete medium. At the phase of exponential growth, the medium was replaced with serum free DMEM supplemented with HMME (0, 8, 16, 32, or 64 mM) and cells were cultured for 2 h in the dark in a tissue-culture incubator at 37â„ƒ and 5% CO2. Cells were illuminated with a 632.8 nm He-Ne laser, and the beam was expanded to give a collimated source of 9 mm in diameter. The intensity of the illuminated radiation at the surface of culture plate was adjusted to 14 mW/cm2. Various light doses (0, 1.4, 2.8, 5.6, 11.2 J/cm2 ) were used for PDT treatment at room temperature. The optimized parameters for HMME-PDT were determined by orthogonal design. After light exposure, cells were returned to a tissue culture incubator. Cell's viability was analyzed using a MTT assay 24 h post-irradiation. Viability (%) was expressed as ODsample/ODcontrol -100 %.
Cells were randomly divided into various experimental groups. The beam of He-Ne laser was expanded to give a collimated source of 40 mm in diameter, and the power at the surface of culture was adjusted to 3.5 mW/cm2. CHMm cells were treated with HMME plus laser irradiation, HMME (16mM), irradiated (2.8 J/cm2), or mock treatment (without either HMME or irradiation). Subsequent analysis was carried out at 3h, 6 h, 12 h, 24 h and 48 h post-irradiation.
Mitochondrial superoxide dismutase, glutathione peroxidase and malondialdehyde assays
Cells were treated as outlined above. After being washed twice with PBS, mitochondrial was isolated by using a Mitochondria Isolation kit (KeyGen Mitochondria Isolation Kit). Mitochondria (0.3 mg/mL) were analyzed for Superoxide dismutase (SOD) activity, Glutathione peroxidase (GSH-Px) activity and Malondialdehyde (MDA) concentration using specific kit purchased from Nanjing Jiancheng Bioengineering Institute. The examination of SOD, GSH-Px and MDA were carried out according to the manual of the kit, and the absorbance was detected at 550nm, 412nm and 522nm respectively with a microplate reader.
Staining of mitochondria with heidenhain's iron hematoxylin
Cells were seeded in dishes (35 mm diameter) containing a cover slip at the density of 1-105 cells/dish and cultured for 24 h with complete medium. When cells were in the phase of exponential growth, cells were treated with HMME-PDT accordingly. The slides were then stained with heidenhain's iron hematoxylin, 6 h, 12 h, 24 h, and 48 h following treatment. Briefly, cells were washed twice with PBS, fixed in Stationary Liquid for 30 min, and incubated for 1h in a 3% potassium dichromate solution, and then mordantly dyed in a 5% ammonium ferric sulfate solution at 50â„ƒ for 30 min. The slides were then stained with 10% hematoxylin for 20 min at 50â„ƒ. Mitochondria were cleared by a 2.5% ammonium ferric sulfate solution and turned blue under running water overnight, dehydrated in stepwise in a ethanol gradient, cleared in xylene and finally mounted. Cell was examined by light microscopy.
Assay for mitochondrial membrane potential (Δ Ψm )
To assess the involvement of the damage of mitochondria induced by HMME-PDT, we examined the integrity of the mitochondria. Rhodamine 123 (Rh-123, Sigma-Aldrich Inc., St. Louis, MO, USA) is a fluorescent probe used to monitor ΔΨm. When the electrochemical gradient across the mitochondrial membrane collapses in damaged cells the reagent does not accumulate in the mitochondria and no aggregates form. Cells were treated as described above, followed by trypsinization and staining with 10 μg/mL Rh-123 for 15 min in a tissue culture incubator. Cells were then rinsed with HMME and then cultured in a tissue culture incubator for 60 min, followed by analysis using a FACSCalibur flow cytometer (Becton-Dickinson). The mitochondrial membrane potential measurement was carried out on a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. A total of 10,000 cells per sample were analyzed .
Determination of Ca2+ uptake and release by mitochondria
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Arsenazo III (AIII), a membrane impermeable reagent, is a specific and sensitive Ca2+ probe. By using AIII, Ca2+ movement across the inner mitochondrial membrane can be measured with a dual wavelength spectrophotometer at 20°C following the procedure . Cells treatment and mitochondria isolation were described as outlined above. Briefly, CaCl2 (25 μM) was added to the isolated mitochondria (0.3 mg/mL) for 2 min. AIII (50 μmol/L) and As2O3(10 μM) were then added to measure Ca2+ concentrations in the assay medium based on the absorbance at 685-675 nm to detect the AIII-Ca2+ complex. The changes in the absorbance under different settings will indirectly measure mitochondria-mediated calcium uptake and release .
Assay of the mitochondrial ATPase activities
ATPase activity was determined by the rate of inorganic phosphate released by mitochondria. To prepare mitochondria for this assay, mitochondria were isolated as outlined above. Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and adjusted to 1 mg ml−1. The materials were stored at −70 °C for further use. Na+-K+ ATPase and Ca2+ ATPase activity were measured using commercial kits purchased from the Nanjing Jiancheng Bioengineering Institute according to the manufacturer's procedure.
2.10. Statistical analysis
Data are reported as means ± SD based on at least three independent experiments. Statistical analysis was performed using Student's t test and One-Way ANOVA.P < 0.05 was considered statistically significant.
Characterization of HMME uptake and its distribution in CHMm cells
By taking advantage of HMME emitting green fluorescence light, we have investigated the kinetics of HMME uptake and its cellular distribution in CHMm cells using a fluorescence microscope. HMME uptake was a time-dependent event and the exponential phase of HMME uptake occurred within 10 ~ 60 min (Fig. 1A-E) with the plateau being reached at 60 min. The numbers of cells with readily detectable HMME uptake were at the maximum at 90 min incubation (Fig. 1A-F). The uptake apparently reached the saturation level at 90 minutes, judged on the intensity of fluorescence and the number of fluorescence-positive cells from 90 minute and onward (Fig. 1F-H). HMME can also emit red fluorescence upon excitation by UV (wavelength from 330 to 380nm). By using the photo bleaching technique, the distribution of HMME in CHMm cells has been determined. Following the course of photo bleaching, the intensity of HMME-derived red fluorescence was predominantly decreased in the cytoplasm (Fig. 2), demonstrating that upon its uptake into CHMm cells HMME mainly stayed in the cytosol.
Optimal of HMME-PDT treatment using CHMm cells
For the therapeutic purpose of using HMME-PDT, the goal is to maximize its cytotoxic effect with minimal doses in terms of the amount of HMME and the energy level of laser used. We have thus determined the toxic impact of HMME-PDT in CHMm cells using MTT-based viability assay. While the photosensitizer alone did not affect the survival of CHMm cell in the doses used, laser irradiation exposure only marginally reduced cell survival at the energy levels employed (Fig 3). However, the combination of both robustly reduced CHMm cell viability. The maximal reduction in cell viability was achieved at 16mM HMME and 2.8 J/cm2 laser irradiation. Further increases in either or both did not further enhance the efficacy or the toxicity of the HMME-PDT treatment (P >0.05). Therefore, this condition (16mM HMME plus 2.8 Jâˆ™cm-2 laser irradiation) had the maximal impact on cell viability (p <0.05) (Fig 3).
Effect of HMME-PDT on the mitochondrial SOD, GSH-PX and MDA of CHMm cells
HMME-PDT at the optimized condition reduced mitochondria SOD activity in comparison to the control group (p<0.01) (Table 1). The reduction was timely dependent. Similar trend on mitochondrial GSH-PX was also observed, in which HMME-PDT inhibited GSH-PX in a time-dependent manner (Table 1).
In a reverse manner, HMME-PDT increased mitochondria MDA contents in comparison to the control group (P<0.01) (Table 1). The differences between 3 h and 6 h as well as between12 h and 24 h reached the statistical significant level (p<0.05) (Table.1). In line with the levels of MDA positively reflect the cellular oxidative status, the above observations collectively demonstrate that HMME-PDT enhances ROS accumulation in the mitochondria.
HMME-PDT induces alterations in mitochondrial structure
Our observation that HMME-PDT caused ROS accumulation in the mitochondria suggests that HMME-PDT may cause mitochondrial injuries. To investigate this possibility, CHMm cells were treated with light, HMME, or HMME-PDT for 3, 6, 12, 24 and 48 hours, followed by the examination of mitochondrial morphology.
Mitochondria can be stained as blue granules with heidenhain's iron hematoxylin. The health status and the number of mitochondria can be judged from the intensity and shape of the staining. The staining intensity decreased means that some of the mitochondrial were destructed and disappeared. In comparison to mock-treatment (control) and the treatments of HMME or PDT, the rather homogenous staining was significantly changed in cells treated with HMME-PDT (Fig 4). Following the time course of treatment, not only the intensity of the staining was decreased but also the shape of the staining was changed (Fig 4). In cells treated with HMME-PDT for 24 and 48 hours, shrinkage in both cells and mitochondria was evident (Fig 4H, I). Cells were preloaded with Rh-123 and treated with HMME-PDT, and then the cells were tested using flow cytometry. The Δ Ψ m-sensitive dye, Rh-123 accumulates in the mitochondria with an intact transmembrane potential, forming aggregates with green fluorescence. The results showed that the ΔΨ m decreased dramatically at about 6 h after HMME-PDT and continued to decrease with time ( Fig. 5). Clearly visible changes started at 6 hour treatment, which went on to apoptotic morphology at 24 hour treatment and onward (Fig 4a,b).
HMME-PDT induced mitochondrial dysfunction in CHMm cells
The observation that HMME-PDT altered mitochondrial morphology or structure strongly indicates that HMME-PDT may also disrupt mitochondrial function. As mitochondria play a major role in regulating calcium signaling via calcium uptake and release, we have thus analyzed the impact of HMME-PDT on these processes. In comparison to mitochondria isolated from the control cells, the Ca2+ transportation ability (uptake and release) of mitochondria isolated from cells treated with HMME-PDT was significantly reduced (Fig. 6). The magnitudes of decrease in calcium transportation followed the time course of treatment. At 48 hours treatment, mitochondria virtually lose their calcium transportation (uptake and release) capacity (Fig 6).
In line with the contributions of mitochondrial ATPase activity to the function of mitochondrial respiratory chain and electron transport, we were able to show that HMME-PDT reduced the activities of the mitochondrial Na+-K+ - ATPase and Ca2+-ATPase in CHMm cells (Table 2). The kinetics of reducing ATPase activity followed the time course of HMME-PDT treatment (Table 2).
Collectively, the above results reveal that HMME-PDT significantly compromised mitochondrial ability to transport calcium.
Mammary cancer is the most common malignancy occurring in unspayed female dogs comprising approximately 52% of all neoplasms. The incidence of mammary carcinomas in canines is 3 times that documented in humans . Dogs with poorly differentiated tumors have increased risk of recurrent or metastatic disease within 2 years following mastectomy with a 90% recurrence rate for the most dedifferentiated tumors. Our long-term goal is to find an effective treatment that may be used clinically for the treatment of canine breast cancer. Hematoporphyrin monomethyl ether (HMME) is a promising second-generation porphyrin-related photosensitizer for PDT, and it has already been used clinical trials for PDT of PWS and cancers. Our study has shown that HMME-mediated PDT is very effective in promoting the dysfunction of mitochondrial in cancer cells. So it might develop a promising treatment for canine breast cancer.
Singlet oxygen and reactive oxygen species (ROS) were produced from HMME upon excitation by laser . The subcellular localization of the photosensitizer dictates its potency in causing damages to the cell, because the reactive oxygen could only diffuse within 20 nm in distance owning to its short half-life [22, 23]. HMME was mainly distributed in the cytoplasm, which is consistent with HMME being incapable of passing through the nuclear membrane . In the cytosol, HMME mainly distributes in the mitochondria, endoplasmic reticulum, Golgi, and lysosomes . Substantial amount of evidence suggests that mitochondria play a major role in PDT-induced apoptosis in cancer cells [26-28].
High concentrations of reactive oxygen species are well known to cause oxidative damage to the ultrastructure of mitochondria and thereby disrupt mitochondrial function and cause cell death .The underlying mechanisms can be attributable to HMME-PDT's direct impact on ROS production as well as its indirect effect in which HMME-PDT inhibited mitochondrial reductase (SOD and GSH-PX) activities. The end results are the accumulation of ROS in mitochondria to high levels, which results in structural damages in mitochondria as well as compromising mitochondrial functions.
Under a variety of apoptotic settings, the structure and function of the mitochondrial membrane were altered, which contributed to the release of cytochrome c, apoptosis inducing factor (AIF), and other apoptotic factors from the inter membrane space of the mitochondria into the cytosol [30-33]. Changes in mitochondrial transmembrane potential were the leading cause of interference to the mitochondrial respiratory chain, energy production and cell survival . Mitochondrial membrane lipid peroxidation reduced ability of the mitochondria Ca2 + transporters and mitochondrial ATPase activity, which provides an additional support that HMME-PDT causes mitochondrial dysfunction.
HMME-PDT caused damages of mitochondrial structure and mitochondrial dysfunction, and it led to death of canine breast cancer. Based on the implication of these findings, the clinical application of HMME-PDT and the relevance to the prediction of clinical outcome need to be further studied.
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper
This study was supported entirely by the fund of Supported by Doctoral Tutor Fund of Ministry of Education of China (Grant No.20122325110012).