María C. García Vior

Photodynamic effects of isosteric water-soluble phthalocyanines on human nasopharynx KB carcinoma cells

The photodynamic activity of water-soluble cationic zinc(II) phthalocyanines using human nasopharynx carcinoma (KB cells) was investigated. A sulfur-linked cationic dye, named: 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium)ethylsulfanyl]phthalocyaninatozinc(II) tetraioidide (13) is the most active of four sensitizer assays and shows a singlet oxygen quantum yield of 0.58 and a higher bathochromic shift of 10 nm for the Q-band as compared with the oxygen-linked cationic aliphatic phthalocyanine: 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium)ethoxy]phthalocyaninatozinc(II) tetraioidide (11) and the best photo-stability in water in comparison with their tetra-alpha-substituted counterparts 1,8(11),15(18),22(25)-tetrakis[(2-trimethylammonium)ethoxy]phthalocyaninatozinc(II) tetraioidide (12) and 1,8(11),15(18),22(25)-tetrakis[(2-trimethylammonium)ethylsulfanyl]phthalocyaninatozinc(II) tetraioidide (14). Phthalocyanine 13, partially localized in lysosomes, led to cell photoinactivation in a concentration- and light dose-dependent manner. After photodynamic treatment, compound 13 induced an apoptotic response--as indicated by morphological cell changes--an increase in the activity of caspase-3 and the cleavage of poly-ADP-ribose-polymerase substrate (PARP).

Synthesis and comparative photodynamic properties of two isosteric alkyl substituted zinc(II) phthalocyanines.

The synthesis and photophysical parameters of two novel isosteric cationic zinc(II) phthalocyanines: 2,9(10),16(17),23(24)-tetrakis[(N-butyl-N-methylammoniumethylsulfanyl]phthalocyaninatozinc(II) tetraiodide (6) and 2,9(10),16(17),23(24)-tetrakis[(N-dibutyl-N-methylammonium)ethoxy]phthalocyaninatozinc(II) tetraiodide (7) were investigated. Maximum absorption values were 686.5 nm and 678 nm for 6 and 7, respectively, whereas singlet molecular oxygen generation was 0.42 and 0.67, respectively. The photodynamic effect and the cellular uptake of both phthalocyanines were evaluated on human nasopharynx KB carcinoma cells. After light exposure, phthalocyanine 6 showed a higher cytotoxic activity than 7. In addition, a higher intracellular uptake of 6 and a preferential localization within lysosomes were demonstrated. The production of a greater amount of reactive oxygen species after phthalocyanine 6 irradiation would be responsible for its potent phototoxic action on KB cells.

Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced a caspase-dependent apoptotic response, being activation of caspase-8, -9 and -3 the result of a post-mitochondrial event.

Photodynamic Effects of Zinc(II) Phthalocyanine‐Loaded Polymeric Micelles in Human Nasopharynx KB Carcinoma Cells

A major difficulty in photodynamic therapy is the poor solubility of the photosensitizer (PS) under physiological conditions which correlates with low bioavailability. PS aggregation leads to a decrease in the photodynamic efficiency and a more limited activity in vitro and in vivo. To improve the aqueous solubility and reduce the aggregation of 2,9(10),16(17),23(24)‐tetrakis[(2‐dimethylamino)ethylsulfanyl]phthal‐ocyaninatozinc(II) (Pc9), the encapsulation into four poloxamine polymeric micelles (T304, T904, T1107 and T1307) displaying a broad spectrum of molecular weight and hydrophilic–lipophilic balance was investigated. The aqueous solubility of Pc9 was increased up to 30 times. Morphological evaluation showed the formation of Pc9‐loaded spherical micelles in the nanosize range. UV/Vis and fluorescence studies indicated that Pc9 is less aggregated upon encapsulation in comparison with Pc9 in water–DMSO 2% and remained photostable. Pc9‐loaded micelles generated singlet molecular oxygen in high yields. Photocytotoxicity assays using human nasopharynx KB carcinoma cells confirmed that the encapsulation of Pc9 in T1107 and T1307 increases its photocytotoxicity by 10 times in comparison with the free form in water–DMSO. In addition, Pc9 incorporated into cells was mainly localized in lysosomes.

Phototoxic action of a zinc(II) phthalocyanine encapsulated into poloxamine polymeric micelles in 2D and 3D colon carcinoma cell cultures

Photodynamic therapy is emerging as a hopeful method for the treatment of oncological diseases. In the search of novel therapeutic strategies for colorectal cancer, in this work we reported the photocytotoxic activity of a lipophilic zinc(II) phthalocyanine on a murine colon adenocarcinoma cell line (CT26 cells). The 2,9(10),16(17),23(24) tetrakis[(2-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II), named Pc9, was encapsulated into Tetronic® 1107 polymeric poloxamine micelles (T1107) and assayed in 2D and 3D cell cultures. We showed that the formulation Pc9-T1107 was efficient to reduce cell viability after photodynamic treatment both in 2D cultures (IC50 10±2nM) as well as in CT26 spheroids (IC50 370±11nM). Cellular uptake of Pc9-T1107 was a time- and concentration-dependent process, being the phthalocyanine formulation mainly incorporated into lysosomal vesicles and endoplasmic reticulum cisterns, but not in mitochondria. Pc9-T1107 also induced the formation of reactive oxygen species immediately after cell irradiation. We also found that the phototoxic action of Pc9-T1107 was partially reversed in the presence of antioxidants, such as TROLOX and N-acetyl-cysteine. In addition, we showed that Pc9-T1107 treatment triggered an apoptotic cell death, as suggested by the detection of pyknotic nuclei, the reduction in the expression levels of procaspase-3 and the increase in caspase-3 enzymatic activity.

Lysosomal permeabilization and endoplasmic reticulum stress mediate the apoptotic response induced after photoactivation of a lipophilic zinc(II) phthalocyanine

We have previously reported that the phototoxic action of the lipophilic phthalocyanine Pc9 (2,9(10),16(17),23(24) tetrakis[(2-dimethylamino)ethylsulfanyl]phthalocyaninatozinc(II)) encapsulated into poloxamine micelles is related to the induction of an apoptotic response in murine colon CT26 carcinoma cells. In the present study, we explored the intracellular signals contributing to the resulting apoptotic death. We found that Pc9-T1107 arrests cell cycle progression immediately after irradiation promoting then an apoptotic response. Thus, 3 h after irradiation the percentage of hypodiploid cells increased from 5.9 ± 0.6% to 23.1 ± 0.1%; activation of caspases 8 and 9 was evident; the population of cells with loss of mitochondrial membrane potential increased from 1.1 ± 0.4% to 44.0 ± 9.3%; the full-length forms of Bid and PARP-1 were cleaved; and a 50% decrease of the expression levels of the anti-apoptotic proteins Bcl-2 and Bcl-XL was detected. We also found that the photosensitizer, mainly retained in lysosomes and endoplasmic reticulum (ER), promotes the permeabilization of lysosomal membranes and induces ER stress. Lysosomal membrane permeabilization was demonstrated by the reduction of acridine orange lysosome fluorescence, the release of Cathepsin D into the cytosol and ∼50% decrease of Hsp70, a chaperone recognized as a lysosomal stabilizer. Cathepsin D also contributed to Bid cleavage and caspase 8 activation. The oxidative damage to the ER induced an unfolded protein response characterized, 3 h after irradiation, by a 3-fold increase in cytosolic Ca2+ levels and 3-4 times higher expression of ER chaperones GRP78/BIP, calnexin, Hsp90 and Hsp110. The cell death signaling promoted by cytosolic Ca2+, calpains and lysosomal proteases was partially abolished by the Ca2+ chelator BAPTA-AM, the calpain inhibitor PD 150606 and proteases inhibitors. Furthermore, Bax down-regulation observed in Pc9-treated cells was undetectable in the presence of PD 150606, indicating that calpains contribute to Bax proteolytic damage. In summary, our results indicate that photoactivation of Pc9-T1107 led to lysosomal membrane permeabilization, induction of ER stress and activation of a caspase-dependent apoptotic cell death.