a goal for more effective photodynamic therapy of cancer
Two combined photosensitizers: a goal for more effective photodynamic therapy of cancer1Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Madrid 28049, SpainReceived 28 October 2013; Revised 15 January 2014; Accepted 16 January 2014
Edited by A Stephanou
Top of pageAbstractPhotodynamic therapy (PDT) is a clinically approved therapeutic modality for the treatment of diseases characterized by uncontrolled cell proliferation, mainly cancer. It involves the selective uptake of a photosensitizer (PS) by neoplastic tissue, which is able to produce reactive oxygen species upon irradiation with light, leading to tumor regression. Here a synergistic cell photoinactivation is reported based on the simultaneous administration of two PSs, zinc(II) phthalocyanine (ZnPc) and the cationic porphyrin meso tetrakis(4 N methylpyridyl)porphine (TMPyP) in three cell lines (HeLa, HaCaT and MCF 7), using very low doses of PDT. Analysis of changes in cytoskeleton components (microtubules and F actin), FAK protein, as well as time lapse video microscopy evidenced that HeLa cells were induced to undergo apoptosis, without losing adhesion to the substrate. Moreover, 24 after intravenous injection into tumor bearing mice, ZnPc and TMPyP were preferentially accumulated in the tumor area. PDT with combined treatment produced significant retardation of tumor growth. We believe that this combined and highly efficient strategy (two PSs) may provide synergistic curative rates regarding conventional photodynamic treatments (with one PS alone).
Photodynamic therapy (PDT) is a multi step and successful clinically approved oncologic therapeutic modality, which involves the selective uptake of a photosensitizer (PS) by neoplastic tissue followed by illumination with light of appropriate wavelength that is able to trigger photochemical reactions that lead to the generation of reactive oxygen species (ROS), mainly singlet oxygen (1O2), which result in tumor regression. PDT based antitumor effects are multifactorial and include (i) direct killing of tumor cells, (ii) damage to the vasculature, and (iii) triggering of an antitumor immune response.1, 2, 3 PDT has been approved in several countries to treat a variety of cancers, such as skin, bladder, lung, esophagus, and cervix among others.2
PDT can be used in combination with a variety of currently used cancer therapies, including chemotherapy,4, 5 radiation therapy,6 surgery,7 gene therapy,8 and immunotherapy,9 without compromising these therapeutic modalities. Moreover, the adverse effects of chemotherapy or radiotherapy are absent, and considering its unique 1O2 dependent cytotoxic effects PDT can be safely combined without the risk of inducing cross resistance.
Combined strategies have been introduced for cancer treatments, including PDT with tumor suppressors,10, 11 inhibitors,12 and anti angiogenic drugs.2 Another way to enhance PDT efficacy involves an increase in PS delivery and specificity through conjugation to tumor targeting molecules. PS encapsulation into nanoparticles or combining PDT with agents that target signal transduction pathways, seems to increase efficacy and selectivity of PDT.13, 14, 15 However, there are few studies that have attempted to determine the effects of the combination of two PSs as a new strategy.2, 16, 17, 18
In this study, we explored the photosensitized effects of PDT mediated by simultaneous administration of two PSs: zinc(II) phthalocyanine (ZnPc) and the cationic porphyrin meso tetrakis(4 N methylpyridyl)porphine (TMPyP), in different tumor cell lines as well as in tumor bearing mice (preliminary results). Likewise, irradiation alone did not induce cytotoxicity (data not shown). Figure 1 shows changes in cell viability caused by different treatments. Photodynamic treatments with each PS alone did not significantly affect HeLa cell survival, at both 24 and 48 after treatments. Combined treatment produces highly significant effects on the survival of the three cell lines used. Data correspond to mean values from at least six different experiments. On the other hand, MCF 7 cells showed higher photosensitization at 24 It is important to note that 48 after photodynamic treatments with each PS alone, surviving fractions of both cell lines, HaCaT and MCF 7, increased until they attained similar values as described for control cells, but in the case of combined treatment we observed a decrease in cell viability, which confirmed a high inactivation efficiency of our combined strategy (see Figure 1b).
Statistical evaluation (one way ANOVA Tukey test) showed that the PDT effect in combination treated HeLa cells at 24 and 48 differs significantly from control, ZnPc alone and TMPyP alone treated cells (P in all cases). Cell viability differences between other groups were not significant at any time interval. In case of HaCaT cells, highly significant viability differences were observed between control, ZnPc or TMPyP versus combination treated cells (P at both post treatment times. In ZnPc treated HaCaT cells, weak significant differences were detected at 24 but not at 48 in comparison with control cells (P In MCF 7 cells, significant differences in cell viability were observed for the TMPyP group (P and the ZnPc group (P in comparison with controls at 24 which were not significant at 48 Highly significant differences were detected between the combination treated group and all other groups (P at both time intervals. Well known morphological criteria26 were used for identification of apoptotic and necrotic cells.
Figure 2.(A) Morphology of HeLa cells in phase contrast or DIC and after NR or H 33258 staining. (a Control cells. Note the increasing amount of cells with clear apoptotic morphology (cell shrinkage and chromatin fragmentation). Note the homogeneous nuclear condensation and giant bubbles characteristic of necrosis. Values are mean of three independent determinations
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HeLa cells treated with one PS alone or only irradiated presented similar morphology to control cells (data not shown). However, after photodynamic treatment with ZnPc HeLa cells showed significant morphological changes, depending on the time elapsed after the end of treatment (3, 6, and 24 and on light dose. As shown in Figure 2Ab, a large number of HeLa cells have undergone apoptosis 3 after photodynamic treatment, as deduced from cell shrinkage, chromatin condensation, and nuclear fragmentation, which are typical apoptotic features. At 6 (Figure 2Ac) and 24 (data not shown), most cells have undergone apoptosis and only few cells remain alive. Also, uniform chromatin condensation leading to pyknotic nuclei was detected. Twenty four hours after treatment, giant bubbles were broken and cytoplasm remnants were found still attached to the culture substrate (data not shown). As can be seen in Supplementary Figure 1A, 24 after phototreatments, HeLa cells preincubated with TMPyP DPPC showed similar morphology to TMPyP treated cells, and we confirmed that this toxicity was not due to DPPC mixture alone (no ZnPc). Thus, it seems unlikely any interaction of cationic porphyrin with DPPC liposomes. Furthermore, we analyzed whether the mechanism of entry could be altered when both PSs were administered together. We performed incubations of HeLa cells for 1 with combined PSs at 4 commonly used for endocytosis inhibition. Cells only irradiated presented similar morphology to control cells (data not shown). As can be seen in Supplementary Figure 2, while 3 after ZnPc PDT or TMPyP PDT treatment (in case of MCF 7 cells, or only after ZnPc PDT in HaCaT cells) a slight increase in number of metaphase cells could be detected (with chromosomes properly aligned in the equatorial plate), 48 after individual photodynamic treatments there were no signs of toxicity (data not shown). On the contrary, combined PDT induced deep morphological changes characteristic of an apoptotic death, depending on time elapsed after the end of treatment in both cell lines. Therefore, these results confirm that synergistic effect, with most cell death induced by apoptosis, is not exclusive of the HeLa cell line.
Electron microscopy studies Micrographs taken by scanning electron microscopy (SEM) confirmed previous results. Interphase control cell morphology (Figure 3Aa) was flattened as well as polygonal, and cell surface showed numerous connections between plasma membrane of neighboring cells. However, 3 after apoptotic treatment (Figure 3Ab), almost all cells were shrunken, had numerous vesicles and showed prolongations attached to the substrate. Six hours later (Figure 3Ac), cells showed typical apoptotic morphology with deformations of membrane as and loss of intercellular connections. At 24 only apoptotic cell debris were observed (data not shown). HeLa cells started the process with an immediate and massive production of small surface evaginations (bubbles), but without membrane disruption. A few minutes later, these surface deformations converged into a big, single bubble. Particularly, cells observed by SEM after 6 of this treatment (Figure 3Ad) exhibited plasma membrane rupture after detachment of the giant bubble, with gradual liberation of cytoplasmic content.
Figure 3.(A) Morphological changes of HeLa cells incubated 1 with both PSs and subjected to red light irradiation, visualized by scanning electron microscopy (SEM). (a) Interphase control cell. (a) Untreated (control) cells. In the cytoplasm we observed mitochondria, narrow profiles of endoplasmic reticulum, and Golgi apparatus with flattered overlapping cisternae. At 3 and 6 after irradiation, combination treated cells appeared with typical apoptotic morphology (Figures 3Bb and Bc). Three hours after necrotic treatment, membranous cell components were swollen and optically empty, although no evident nuclear damage could be detected. At 6 cells showed an extensive number of vesicles, with no clearly distinguishable organelles and strongly compacted chromatin masses (Figure 3Bd). Especially 18 after treatment, cells hypofluorescence increased to 79.3 compared with controls (1.2 Annexin V assay results obtained for Hela cells are shown in Figure 4B. Figure 4D shows that cleavage of poly (ADP ribose) polymerase (PARP) was visible only when cells were treated with combined PDT.
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