A. T. P. C. Gomesab,
M. A. F. Faustinoa,
M. G. P. M. S. Neves*a,
V. F. Ferreirac,
A. Juarranzb,
J. A. S. Cavaleiroa and
F. Sanz-Rodríguez*b
aQOPNA & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail: gneves@ua.pt
bDepartamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain. E-mail: francisco.sanz@uam.es
cDepartamento de Química Orgânica, Universidade Federal Fluminense, 24020-150, Niterói, RJ, Brazil
First published on 1st April 2015
The photodynamic effect of glycochlorins 1–4 was evaluated for the first time in human epithelial cells (HeLa) and their PDT efficiency was compared with the results obtained when using human normal skin keratinocytes cells (HaCaT). It was demonstrated that the galactose conjugate is the only photosensitizer that is selective for HeLa cells, without causing a significant effect on the HaCaT cells. Morphological studies carried out after the photodynamic treatment attested such selectivity and showed that HeLa cells suffer a strong cytoplasmic vacuolization and a pronounced retraction, while in HaCaT cells the damage, under the same protocol conditions, is scarce.
In general, cancer treatment includes surgery, chemotherapy and radiotherapy. Some of these approaches can cause some physical damage in patients and sometimes the disease is only put in a temporary state of latency.1 Considering this, it is crucial to look for the development of therapeutic approaches that allow the eradication of cancer, with minor damages to patients. The photosensitizing ability demonstrated by porphyrins and analogues in photodynamic therapy (PDT) makes these tetrapyrrolic macrocycles special candidates to be applied on this type of cancer therapy.2 In PDT, the combination of the photosensitizing drug (PS), oxygen and visible light produces lethal cytotoxic agents (e.g. singlet oxygen (1O2) and/or other reactive species) that are responsible for the destruction of malignant cells.3 This therapeutic approach presents as major advantage the fact that the selected PS is non-toxic in the absence of light and also it is a localized treatment with low accumulation in non-specific tissues. Additionally, it is a treatment confined to the irradiated area and can be repeated without the risk of harming neighbouring healthy tissue, since the effect of the non-ionizing activating light on tissues, in the absence of the PS drug, is harmless.4 The clinical interest in formulations based on tetrapyrrolic macrocycles or on their precursors for PDT is demonstrated by health agency approvals of Photofrin®, Levulan®, Visudyne®, Foscan®, Metvix® and Photosense® in US and other countries.5
One of the major challenges of PDT is to promote the selective PS localization on tumour cells in order to increase the efficiency of the neoplastic tissue damage when compared to the normal one.6 One of the approaches to reach this selectivity is based on the synthesis of porphyrins or analogues bearing targeting moieties like carbohydrate ones.7 There are many examples in literature involving the synthesis of carbohydrate–porphyrin conjugates based on the concept that carbohydrate moieties have the ability to target carbohydrate-recognized proteins, which are known for their high expression in certain tumours.3,7–9 Moreover, the conjugation of carbohydrates to porphyrin derivatives can improve their water solubility and bring better hydrophilic/hydrophobic ratios, this being very important in biodistribution and pharmacokinetic profile.3,10 Since another important requirement for an ideal PS is related with the presence of a strong absorption in the visible region near or above 650 nm, where the light penetration in tissues is higher, it was decided to evaluate the photosensitizing ability of glycochlorins containing D-glucose (1), D-ribose (2), D-fructose (3) and D-galactose (4) units (Fig. 1) towards HeLa cells.
The synthesis of theses derivatives was previously reported by us and involved the reaction of meso-tetrakis(pentafluorophenyl)porphyrinatozinc(II) with the corresponding di-acetonides of carbohydrate-substituted α-diazoacetates 5a–d, in the presence of catalytic amounts of CuCl (Scheme 1).11 These reactions are selective for the products of mono-cyclopropanation, yielding preferentially the trans isomers 6a–d. It is also shown that the products and their yields depend on the nature of the carbohydrate moiety. Glycochlorins 1–4 were obtained by demetalation and removal of the isopropylidene groups of the carbohydrate moieties of the cyclopropanation products 6a–d. These derivatives, besides their strong absorption band at ca. 660 nm, proved to be good 1O2 producers.11 In this work, we report for the first time the photodynamic effect of glycochlorin-conjugates 1–4 in a cervical cancer human cell line (HeLa) and in an human normal skin keratinocytes cell line (HaCaT).
The efficiency of these glycochlorin conjugates was assessed by comparing the photodynamic effect in HeLa and in HaCaT cell lines. Both cell types are of epithelial origin and are considered to be an excellent comparative model between non-tumoral vs. tumoral studies in vitro. The photodynamic assays were performed with the glycochlorin conjugates incorporated in liposomal suspensions in order to avoid their aggregation in water. In fact, liposomes are considered valuable carriers and delivery systems for PDT. These vesicles composed by phospholipids have high loading capacity and flexibility to accommodate and deliver PSs with variable physicochemical properties.12 It has been also demonstrated that the PDT outcome of liposomal PS is advantageous, when compared with the non-liposomal PS; a possible explanation is that a liposomal formulation can substantially decrease the extent of PS aggregation.12
Conjugate | Conc. (M) | Irradiation time (min) | Survival fraction (% ± SD) | |
---|---|---|---|---|
HaCaT | HeLa | |||
a Each point corresponds to the mean value ± SD from 3 independent experiments with 4 replicates for each experimental condition. | ||||
1 | 1.0 × 10−7 | 5 | 96.87 ± 1.50 | 113.25 ± 1.17 |
10 | 89.53 ± 1.94 | 101.65 ± 3.74 | ||
20 | 85.37 ± 1.58 | 102.50 ± 1.44 | ||
1.0 × 10−6 | 5 | 63.98 ± 3.33 | 42.53 ± 1.73 | |
10 | 32.80 ± 0.71 | 23.37 ± 5.51 | ||
20 | 15.43 ± 1.74 | 16.29 ± 0.69 | ||
3 | 1.0 × 10−7 | 5 | 70.68 ± 1.36 | 89.44 ± 0.89 |
10 | 65.75 ± 4.42 | 84.57 ± 2.04 | ||
20 | 49.97 ± 4.26 | 60.52 ± 2.61 | ||
1.0 × 10−6 | 5 | 25.93 ± 3.48 | 14.67 ± 0.90 | |
10 | 21.26 ± 0.19 | 13.50 ± 0.15 | ||
20 | 22.87 ± 0.03 | 13.70 ± 0.37 | ||
4 | 1.0 × 10−7 | 5 | 102.82 ± 1.40 | 93.72 ± 9.84 |
10 | 102.60 ± 5.16 | 91.44 ± 6.85 | ||
20 | 106.45 ± 2.37 | 78.68 ± 2.05 | ||
1.0 × 10−6 | 5 | 90.33 ± 9.42 | 62.12 ± 2.05 | |
10 | 82.14 ± 9.05 | 34.65 ± 3.58 | ||
20 | 72.86 ± 3.85 | 30.52 ± 2.06 |
The results show that the photodynamic effect of glycochlorins 1, 3 and 4 on the two epithelial cell lines was dependent on the sugar unit, concentration and irradiation time. With the glucose conjugate 1 the highest decrease in cell survival for both cell lines was achieved at the highest concentration and irradiation time. Additionally, it is possible to observe that the photodynamic effect of this conjugate on non tumoral HaCaT cells is similar or even more significant than the one observed for HeLa cells, principally when the low concentration was tested. These results indicate clearly that glycochlorin 1, under the same experimental conditions, was not selective for tumoral cells, causing a similar photodynamic effect in both HeLa and HaCaT cells.
When the fructose conjugate 3 was used as photosensitizer, the highest decrease in cell survival was also attained at the highest concentration tested. In the case of the tumoral cell line HeLa, at the lowest concentration (1.0 × 10−7 M) and after 20 min of irradiation it is observed a decrease of approximately 40% on cell survival while at the highest concentration (1.0 × 10−6 M) and with the same irradiation time a decrease of 86% is attained. Although this value seems to be, in a first analysis, a very satisfactory reduction in the viability of the tumoral cell line (HeLa) when is compared with the one obtained in the non-tumoral cell line HaCaT, at the same experimental conditions, no significant selectivity was observed. So, in this case the presence of fructose moiety did not improve the chlorin selectivity on HeLa cells.
However, the results summarized in Table 1 show that the photodynamic profile of galactose–chlorin 4 in both cell lines is completely different and very promising when compared with the previous data. In fact, at the lower concentration (1.0 × 10−7 M) and after 20 min of irradiation, this photosensitizer is able to cause a decrease of approximately 21% on the survival of HeLa cells without affecting HaCaT cell line. This selectivity is maintained when the PDT experiments were performed in the presence of this glycochlorin at the highest concentration (1.0 × 10−6 M) for all irradiation periods. For instance, after 20 min of irradiation, the decrease observed in the survival of the HeLa cells was approximately 70%, while the decrease in the survival of HaCaT cells under these conditions of treatment was approximately 27%. This result clearly indicates that this photosensitizer presented a high selectivity for tumoral HeLa cell lines without significantly affecting the viability of non-tumoral HaCaT cell line.
Based on the results obtained and on the idea that a good photosensitizer must possess high selectivity for tumoral cells, we can consider that conjugate 4 is the best candidate to be used as photosensitizer in PDT. The low selectivity of glycochlorins 1 and 3 towards the tumour cell line (HeLa), causing a similar cytotoxic effect in both non-tumoral and tumoral cells, precludes their use as PS. On the other hand, glycochlorin 4 showed high efficiency in photoinactivation of tumoral HeLa cells, causing minor cytotoxic effects on non-tumoral HaCaT cells. Due to this fact, only conjugate 4 was selected to develop further studies.
As it can be observed in Table 2, the survival rates obtained show that chlorin 4 was not toxic to both cell types in the absence of light at the studied concentration, confirming that the cytotoxic effect is due to the reactive oxygen species produced under irradiation.
PS | Conc. (M) | Cell survival (% ± SD) | |
---|---|---|---|
HaCaT | HeLa | ||
a Each point corresponds to the mean value ± SD from 3 independent experiments with 4 replicates for each experimental condition. | |||
4 | 1.0 × 10−7 | 102.35 ± 2.36 | 101.63 ± 4.19 |
1.0 × 10−6 | 108.73 ± 4.90 | 108.05 ± 6.42 |
Morphologically, HaCaT cells tend to form microcolonies with well-developed adhesion systems.18 When this non-tumoral cell line was incubated with conjugate 4 at concentration 1 × 10−7 M, it was possible to verify that the morphological cell changes are scarce and only partial cytoplasmic vacuolization was observed after 20 min of irradiation (Fig. 2). It is important to note that these morphological changes are most apparent at the edge of the colony and no alterations were detected in the centre of the colony. This can be due to the fact that cells located at the edge of the colonies were more exposed to photosensitizer (Fig. 2). It is also clear that this slight cytoplasmic vacuolization does not affect the viability of these cells, since no cytotoxic effect was seen in the MTT experiments.
After similar photodynamic treatment of HeLa cells, it was observed that after 5 min of irradiation the HeLa cells suffer strongly cytoplasmic vacuolization (Fig. 2, inset) that is indicative of cell degeneration processes. After 20 min of irradiation, it was found that the morphology of HeLa cells was dramatically affected, being possible to observe cells presenting a severe cytoplasmic shrinkage as a consequence of the cell death process (Fig. 2, inset). With these experimental conditions, 1.0 × 10−7 M and 20 min of irradiation, it was also possible to observe a reduction in the number of cells present in the preparation (in comparison with the control and after 5 min of irradiation). These results are in agreement with the reduction of 21% of viability found in MTT experiments.
The efficiency of a photosensitizer, both in cell cultures and in tumours, is directly related to its chemical structure, concentration, incubation time, light doses and cellular location.19 A range of cellular components have been described as targets of cytotoxic oxygen species formed during the photodynamic process, such as mitochondria, lysosomes, Golgi apparatus and plasma membrane. Photosensitizers that are located in mitochondria and lysosomes are highly efficient in cell photoinactivation, causing necrosis and apoptosis.20
Having in mind the importance of this fact, we decided to carry out studies of subcellular localization with the most effective and selective photosensitizer, conjugate 4, at the concentration of 1.0 × 10−7 M, the same concentration used for morphological studies. Thus, the cell lines were incubated with the conjugate 4 at 1.0 × 10−7 M for 2 (data not shown), 4, and 6 h in the dark. After each incubation time, cells were washed with PBS in order to remove the photosensitizer that has not been incorporated into cells. After that, coverslips were mounted with PBS and the preparations were observed under blue light in a fluorescence microscope. Two controls were also performed where each cell line were incubated without conjugate 4. The results are presented in Fig. 3.
The distribution pattern of this conjugate was similar in HaCaT and HeLa cells. In both cell types the conjugate 4 was mainly located in granules that closely resemble endosomes/lysosomes (Fig. 3, yellow arrows) since this fluorescent granular signal was similar to that observed after cell incubation with lysotracker. Some larger red fluorescent granules are also seen in the cytoplasm; these granules may correspond to other types of vesicles (white arrows).
As discussed above, HaCaT cells tend to form microcolonies where the cells are very attached to others.18 So, it is important to take into account that the location of the photosensitizer is always at the edge of colony of this cell line and it is only possible to find it within the colony when it is formed by a low number of cells.
In the present study we show how photodynamic therapy with these glycochlorin conjugate affects cultured HeLa tumoral cells. HeLa cells are regarded as one of the best-known models of cell lines lacking functional p53 protein, due to activity of the host HPV16 viruses and to mutations in p53, which occur in over 50% of human tumours.22 On the other hand, HaCaT cells are a transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal and has a transformed phenotype in vitro but it remains non-tumorigenic, therefore this cell line is a good model of non-tumoral cells.18 Using these two in vitro models of HaCaT and HeLa cells, we demonstrate high efficiency of PDT killing tumoral cells when our glycochlorin conjugates were applied.
However, not all compounds showed high selectivity for tumoral cells. First studies on cell viability after PDT, demonstrated that glucose- and fructose-chlorins conjugates 1 and 3 have no cell selectivity, inactivating equally the non-tumoral HaCaT cells and the tumoral HeLa cells. A different situation was observed with the galactose–chlorin (conjugate 4) that proved to be selective for the tumour cell line, inhibiting the growth of HeLa cells without affecting significantly the normal cell line (HaCaT). Interestingly the role of galactose in other PS (porphyrins, chlorins, corroles and phthalocyanines) was already highlighted.23–27
Tumour cells exhibit high levels of glycolysis, despite the presence of oxygen in a phenomenon termed aerobic glycolysis. During the last years it has been hypothesized that targeting glucose metabolism may provide a selective mechanism to kill cancer cells.28 However, glucose (and consequently fructose) is also metabolized in healthy cells through glycolysis, therefore it is expected that HaCaT and HeLa cells recognize equally glucose–chlorin 1 and fructose–chlorin 3.
The action of a PS in the presence of light is in general directly related to the 1O2 production. So, the lack of photodynamic effect in the absence of light can provide useful information about the mechanism that is responsible for the decrease of cellular viability of the studied cells.15,16 In this study it was confirmed that galactose–chlorin 4 is not toxic in the absence of light confirming that the principal mechanism of action is related to the 1O2 production.
Considering the morphological alterations, after the photodynamic treatment with conjugate 4 at concentration of 1.0 × 10−7 M, it was observed that HeLa cells suffer strongly cytoplasmic vacuolization and pronounced retraction, whereas in HaCaT cells the damage observed is scarce, showing only partial cytoplasmic vacuolization of the cells located at the edge of the colonies. These results strongly corroborate the high selectivity of galactose–chlorin 4 for the tumoral cells.
At the same conditions, the subcellular localization studies showed that conjugate 4 localizes in granules that closely resemble endosomes/lysosomes both in HeLa cells and in HaCaT. However, in the case of non-tumoral cell lines this conjugate is always present at the edge of the colony. This specific localization of conjugate 4 may be related to the composition of the liposome formulation used in the administration of the PSs. In fact, the liposome formulations can modulate the intracellular localization of PSs by controlling the interaction of liposomes with the subcellular membrane.12,29 The liberation of PSs from liposomes can be affected by the structure of liposome, temperature, pH, etc. The DPPC liposomes used in this work are pH-sensitive and it is known that they destabilize between pH 5 and 6.3. Since the endosomal and lysosomal pH as well the interstitial pH in tumour tissue is lower than the normal physiological pH,12,29 the release of these liposomes content in cytoplasm of tumoral cells is more effective than in non-tumoral cells.
In conclusion, these promising results on the selectivity demonstrated by galactose–chlorin conjugate towards HeLa cell line will lead us to perform further studies on the mechanism of action of this conjugate.
To assess the possible cytotoxic effect of glycochlorin 4, cells were incubated in the presence of this PS, under the same conditions as for the PDT experiments, but without performing irradiation with red light. Cell viability was also estimated using the MTT colorimetric method. All results obtained represents three independent experiments with replicates.
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