Abstract
Background: Photodynamic therapy (PDT) is linked with oxidative damage of biomolecules causing significant impairment of essential cellular functions that lead to cell death. It is the reason why photodynamic therapy has found application in treatment of different oncological, cardiovascular, skin and eye diseases. Efficacy of PDT depends on combined action of three components; sensitizer, light and oxygen. In the present study, we examined whether higher partial pressure of oxygen increases lethality in HeLa cell lines exposed to light in the presence of chloraluminium phthalocyanine disulfonate (ClAlPcS2). Methods: ClAlPcS2- sensitized HeLa cells incubated under different oxygen conditions were exposed to PDT. Production of singlet oxygen (1O2) and other forms of reactive oxygen species (ROS) as well as changes in mitochondrial membrane potential were determined by appropriately sensitive fluorescence probes. The effect of PDT on HeLa cell viability under different oxygen conditions was quantified using the standard methylthiazol tetrazolium (MTT) test. Results: At the highest oxygen concentration of 28±2 mg/l HeLa cells were significantly more sensitive to light-activated ClAlPcS2 (EC50=0.29±0.05 μM) in comparison to cells incubated at lower oxygen concentrations of 8±0.5 and 0.5±0.1 mg/l, where the half maximal effective concentration was 0.42±0.06 μM and 0.94±0.14 μM, respectively. Moreover, we found that the higher presence of oxygen is accompanied with higher production of singlet oxygen, a higher rate of type II photodynamic reactions, and a significant drop in the mitochondrial membrane potential. Conclusion: These results demonstrate that the photodynamic effect in cervical cancer cells utilizing ClAlPcS2 significantly depends on oxygen level.
Abbreviations: 1O2, singlet oxygen; ClAlPcS2, chloraluminium phthalocyanine disulfonate; CM-H2DCFDA, 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate; DMEM, Dullbecco's modified Eagle's medium; DMSO, dimethylsulfooxide; HeLa, human cervical cancer cell line; JC-1, 5,5’,6,6’- tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine chloride; LED, light-emitting diode; MMP, mitochondrial membrane potential; MTT, methylthiazol tetrazolium bromide; PBS, phosphate-buffered saline; PBS-G, PBS supplemented with 5 mM glucose; PDT, photodynamic therapy; pO2, partial pressure of oxygen; ROS, reactive oxygen species; SOSG, singlet oxygen sensor green reagent.
Photodynamic therapy (PDT) is an alternative treatment modality for malignant neoplasms (primarily as the adjuvant or palliative therapy), but also of non-cancerous diseases including skin and eye conditions (1).
The principal of PDT is the selective accumulation of the drug called sensitizer (photosensitizer, photosensibilizator) inside the sensitive cells and irradiation of the cells by light, which corresponds to the sensitizer's absorption maximum. Following the absorption of photons, the sensitizer transforms from its ground singlet state into an excited singlet state, which can either decay back to its ground state by emitting fluorescence, or can be transformed via intersystem crossing into a relatively long-lived excited triplet state. This can either form free radicals or radical ions that interact with surrounding molecules producing superoxide anion radicals, hydrogen peroxides and hydroxyl radical (type I reaction). Alternatively, the excited sensitizer can directly transfer its energy to oxygen to form the non-radical but highly reactive singlet oxygen (type II reaction). Both reactions can occur simultaneously and the ratio between them depends on the sensitizer and the nature of the substrate molecules (2, 3). In case of excess oxygen, type II reaction prevails. Its product, singlet oxygen, is very reactive and immediately (during tens of nanoseconds) oxidizes neighboring biomolecules, such as proteins, lipids and DNA, resulting in their dysfunction.
Nowadays, it is accepted that high levels of ROS lead to increased cell death, whereas low ROS levels have an effect in promoting tumorigenesis by activating the signaling pathways that regulate proliferation, angiogenesis and metastasis (4, 5). Thus, the higher partial pressure of oxygen (pO2) in tumor tissue is crucial for the effectiveness of PDT. This has been reported in many clinical trials and experimental studies looking at the efficacy of hyperbaric oxygen combined with PDT. Maier and his colleagues presented an enhancement of PDT in patients with advanced esophageal carcinoma when it was carried out under hyperbaric oxygen (6). In an experimental animal model, Jirsa et al. (7) studied the influence of HBO and PDT in tumor-bearing nude mice. They concluded that combining HBO and PDT improves the efficiency of PDT by increasing the depth of tumor cell damage and/or by reducing the doses of sensitizers. On the other hand, another study aimed at human squamous carcinoma cells did not show any significant enhancement in phototoxicity induced by protoporphyrin IX precursors at higher oxygen concentrations (8).
In the present study we investigated the efficacy of PDT on HeLa cell lines using chloraluminium phthalocyanine disulfonate (ClAlPcS2) at different oxygen levels.
Materials and Methods
Cell line, sensitizer and irradiation conditions. The HeLa human cervical cancer cells (1x105 per microplate well) were grown in Dullbecco's modified Eagle's medium (DMEM) containing 0 (control), 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM ClAlPcS2 and stored in a thermobox at 37°C and 5% of CO2 for 24 h. After incubation, DMEM medium was replaced by PBS buffer with 5 mM glucose (PBS-G) at different partial pressures of oxygen related to dissolved oxygen concentrations of 0.5±0.1, 8±0.5, 28±2 mg/l (measured by oxymeter Greisinger 3630, Germany). The lowest or highest oxygen concentration was achieved by pure nitrogen or oxygen bubbling in PBS-G for 15 min. The middle value (8±0.5 mg/l) reflects oxygen solubility in buffer under normal atmospheric conditions. After 30 min of incubation, the cells underwent photodynamic treatment by light emitting diodes (LED) source emitting 660±15 nm light for 3 min at 20 mW/cm2 (i.e. 3.6 J/cm2). To keep appropriate oxygen levels in media, all experiments were carried out under normobaric conditions in a hermetic chamber filled with nitrogen, air, and oxygen.
Estimation of the type I photodynamic reaction products (hydrogen peroxide and its downstream products). After cell incubation in 96-well microplates and DMEM replacement by PBS-G with different oxygen concentration, fluorescence probe 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Molecular Probes by Life Technologies) was added at the final concentration of 10 μM and left incubated for 20 min in the dark. The assay using CM-H2DCFDA is especially sensitive to the increased production of hydrogen peroxide or some of its downstream products (9). Upon crossing the membrane, the compound undergoes deacetylation by intracellular esterases producing the non-fluorescent CM-H2DCF, which quantitatively reacts with above mentioned oxygen species inside the cell to produce the highly fluorescent dye CM-DCF. Immediately after PDT, fluorescence of CM-DCF within sensitized cells (excitation and emission wavelength was 480 nm and 530 nm, respectively) was recorded by the fluorescence reader Tecan Infinite 200pro (Tecan Group, Switzerland).
Estimation of the type II photodynamic reaction product (singlet oxygen). Another fluorescence probe, singlet oxygen sensor green reagent (SOSG, Molecular Probes by Life Technologies) was used to detect production of singlet oxygen in HeLa cells accompanying PDT. The final concentration in each microplate well was 2 μM. Fluorescence emission intensity at 530 nm was measured at an excitation wavelength of 500 nm.
Mitochondrial membrane potential. Mitochondrial membrane potential was evaluated by the fluorescence probe 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine chloride (JC-1, Biotium). JC-1 is a mitochondrial dye that stains mitochondria in living cells in a membrane potential-dependent fashion. JC-1 monomer is in equilibrium with so-called J-aggregates, which are favored at higher dye concentration or higher mitochondrial membrane potential. The monomer JC-1 is characterized by green fluorescence (emission at 530 nm), while the J-aggregates have red fluorescence (emission at 590 nm). At the beginning HeLa cells underwent PDT as was described in the first paragraph of this chapter and subsequently incubated with JC-1 at the final assay concentration of 2 μg/l in the hermetic chamber for 20 min. After that incubation, fluorescence emission intensity at 530 nm and 590 nm was measured at an excitation wavelength of 490 nm.
MTT viability test. After light exposition, a buffer in each microplate well was replaced by fresh DMEM, and survival cells were cultivated under standard incubation conditions (37°C, 5% of CO2) for 6 h. After 6 h of incubation in the presence of 0.5 mg/ml MTT, the growth medium was replaced by 100 μl of DMSO to dissolve the formazan crystals. Concentration of formazan reflecting viability of cells was spectrophotometrically evaluated by measuring its absorbance at 570 nm.
Data analysis. Data shown illustrate means±standard errors for 6 independent experiments. The one-way analysis of variance (ANOVA) was used for comparisons between experimental groups. Significance was set at p<0.05.
Results
Determination of ROS production. The effect of different concentrations of ClAlPcS2 and oxygen on ROS formation in HeLa cells was immediately investigated after a 3 min application of PDT. Production of 1O2, H2O2 and its downstream products was determined using appropriately sensitive fluorescence probes SOSG and CM-H2DCFDA, respectively (Figures 1, 2 and 3). The obtained results showed significant differences in ROS production between cells exposed to low (0.5±0.1 mg/l) and high (28±2 mg/l) concentration of oxygen. While the presence of higher oxygen concentration led to a higher yield of the 1O2, at lower oxygen levels, an increased production of H2O2 and its downstream products was observed.
Dependence of 1O2 production in HeLa cells on concentration of ClAlPcS2 during a 3 min irradiation with a light intensity of 20 mW/cm2 and on oxygen concentration of 0.5±0.1 mg/l (□) and 28±2 mg/l (○). Each value represents mean±S.E. from 6 independent experiments. *Significant difference compared to different oxygen concentration (p<0.05).
Dependence of H2O2 production and its downstream products in HeLa cells on concentration of ClAlPcS2 during a 3 min irradiation with a light intensity of 20 mW/cm2 and on oxygen concentration of 0.5±0.1 mg/l (□) and 28±2 mg/l (○). Each value represents mean±S.E. from 6 independent experiments. *Significant difference compared to different oxygen concentration (p<0.05).
Effect of PDT utilizing ClAlPcS2 on mitochondrial membrane potential in HeLa cells. The dependence on ClAlPcS2 concentration at oxygen level of 0.5±0.1 mg/l (circle symbols) and 28±2 mg/l (square symbols) was determined by measuring the fluorescence of JC-1 probe. JC-1 is a mitochondrial dye that stains mitochondria in living cells in a membrane potential-dependent fashion, where JC-1 monomer with a green fluorescence (open symbols) is in equilibrium with red fluorescent J-aggregates (filled symbols), which are favored at higher dye concentration or higher mitochondrial membrane potential. Values represent mean±S.E. from 6 independent experiments.
Determination of MMP. Based on our previously reported results studying photodynamic effects of various sensitizers on human melanoma cells (10), we have examined whether PDT using ClAlPcS2 accompanies changes in MMP also in HeLa cells and whether these changes depend on different levels of oxygen. The MMP assay using JC-1 fluorescence probe showed a significantly higher decrease of MMP in concentration dependent manner of the sensitizer. Moreover the drop in MMP was markedly higher in HeLa cells when they were exposed to higher oxygen concentration (28±2 mg/l). On the other hand, the non-significant changes in the JC-1 monomers fluorescence can reflect the total concentration of the probe in assays (microplate wells) rather than changes in MMP inside HeLa cells. Thus, these results indicate mitochondrial membrane depolarization that can be connected with the early response of PDT on HeLa cells using ClAlPcS2.
MTT viability test. The MTT viability test was used in order to investigate whether photodynamic reaction of the type II producing more 1O2 and accompanying significant drop in MMP is more effective in HeLa cell lines compared to such treatment conditions that give a higher yield of type I reaction. Figure 4 shows a dependence of HeLa cell survival expressed as absorbances of formazan, which is produced only by live cells. In addition, the MTT test proved that the viability was significantly influenced in the ranges of sensitizer concentrations tested. In Table I the EC50 values for the tested sensitizer under different oxygen conditions are summarized. A significant influence of the sensitizer and oxygen concentrations at an irradiation dose of 3.6 J/cm2 was verified from dose–response curves using software Microcal Origin (OriginLab Corporation, Northampton, USA) (p<0.05, 6 independent samples for each sensitizer concentration). Moreover, the viability test showed that there was no significant lethal effect on HeLa cells when the sensitizer was not activated by light irradiation.
Effect of PDT utilizing ClAlPcS2 on HeLa cell survival at different oxygen levels. EC50 obtained from appropriate dose–response curves acquired by Microcal Origin software is defined as half the maximal effective concentration of the sensitizer where 50% of its maximal effect is observed. Values represent mean±S.E. from 6 independent experiments.
Discussion
Growth of tumors requires a higher supply of oxygen and nutrients. Often due to insufficient vascularization, tumor cells undergo nutrient deprivation, metabolic acidosis, and hypoxia, primarily at the tumor center. Oxygen partial pressure in tumors ranges from 2.5 to 30.0 mmHg (i.e. 0.1 to 1.2 mg/l of dissolved oxygen at 37°C, 0.9% salinity, and normal atmospheric pressure). This is in contrast to normal tissues and the tumor periphery where oxygen partial pressures are between 30 and 60 mmHg (11, 12). Thus, it would seem that lack of oxygen could suppress tumor growth. However, several reports have demonstrated that decreased oxygen partial pressure induces multiple cellular adaptation processes, which sustains and fosters cancer progression, for example through the induction of angiogenesis, changes in metabolism and upregulation of genes involved in cell survival/apoptosis, giving rise to a highly aggressive malignant cell line (13, 14). Therefore, rather than hypoxia, normoxic or hyperoxic conditions in tumor may be better for its treatment. Moreover, there is no evidence indicating that higher pO2 neither acts as a stimulator of tumor growth nor as an enhancer of recurrence (11, 14). Furthermore, there are a few studies that have shown a suppressive effect of hyperbaric oxygen therapy on tumor growth (15) or when it was followed by radiotherapy (16) or chemotherapy (15). Based on several clinical studies that do not show any significant treatment effect of the hyberbaric oxygen on cervical cancer, even if it was used in combination with radiotherapy (11), Moen and Stuhr (14) concluded that cervical cancer does not seem to be a good candidate for that type of therapy. However, we have now provided evidence that photodynamic therapy under hyperoxic conditions may have a different impact.
Dependence of HeLa cell survival on the PDT utilizing different concentrations of ClAlPcS2 at different oxygen level of 0.5±0.1 (▸), 8±0.5 (▪) and 28±2 (●) mg/l. Cell viability was determined by measuring the activity of living cells using the MTT. The increased pO2 and increased sensitizer concentration have a significant effect on cell survival. Furthermore, the data (◂) show no or negligible cytotoxic effect of the sensitizer in the absence of light under normoxia (8±0.5 mg O2 /l). Values represent mean±S.E. from 6 independent experiments.
Photoactivation of any fluorescence substance can lead to photobleaching, photon-induced chemical damage linked with loss of the ability to fluoresce, and also photodynamic effect.
Moan and his coworkers (17, 18) have extensively investigated photodegradation of a number of photosensitisers and have proposed that photobleaching can be mediated by 1O2. However, our results show that EC50 value for ClAlPcS2 in HeLa cells was lower in the presence of a higher level of 1O2, and thus expresses higher potency and most likely does not show photodegradation under our non-laser irradiation condition. On the other hand, Ishii (19) presumes that a degree in photodegradation is required when deeper structures are being treated, because it causes better light penetration. Despite the presence of the highest concentration of the dissolved molecular oxygen, higher production of 1O2 can also be achieved by the use of phthalocyanine complex with a central heavy metal ion (19). However, whether application of the higher partial oxygen pressure or rather use of the heavy atoms in the phthalocyanine sensitizers is better to enhance PDT efficacy in vivo, has yet to be investigated.
When cells are exposed to an atmosphere with reduced oxygen concentration, cells readily response by inducing adaptive reactions for their survival, increase glycolysis, reduce phosphorylation, and activate certain hypoxia-inducible transcription factors when hypoxia is prolonged (20). Oxygen is the terminal acceptor of electrons from cytochrome c oxidase (Complex IV of the mitochondrial respiratory chain). It has been reported that measurements of mitochondrial oxidative phosphorylation in mammalian cells are not dependent on oxygen concentration up to at least 20 μM (i.e. 0.64 mg/l) at pH 7.0 and the oxygen dependence becomes markedly greater as the pH gets more alkaline (21). Our results support this finding since HeLa cells incubated at lower pO2 (0.5±0.1 mg/l) are characterized with a significantly lower value of MMP compared to cells exposed to high pO2 (28±2 mg/l). Moreover the reduction in MMP showed a concentration-dependent manner with the used sensitizer, suggesting that mitochondria can be a sub-cellular target of the PDT using ClAlPcS2 for this type of cancer.
Aknowledgements
I would like to thank Prof. Mauricio da Silva Baptista, Ph.D and Prof. Alicia J. Kowaltowski, MD, Ph.D of University of Sao Paulo in Brazil for not only giving me the opportunity to work in their laboratories during my research visit in 2013 but also for familiarizing me with novel approaches in the photodynamic studies. This work was supported by the grant project CZ.1.07/2.4.00/17.0015, CZ.1.05/3.1.00/14.0307, and CZ.1.07/2.3.00/30.0004.
- Received May 30, 2014.
- Revision received June 5, 2014.
- Accepted June 6, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
