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

Abstract

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-X 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 Ca levels and 3-4 times higher expression of ER chaperones GRP78/BIP, calnexin, Hsp90 and Hsp110. The cell death signaling promoted by cytosolic Ca, calpains and lysosomal proteases was partially abolished by the Ca 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.

Zinc(II) phthalocyanines as photosensitizers for antitumor photodynamic therapy

“…In addition, the reduction of caspase 8 activity observed after incubation of ZnPc-treated with Pepstatin A supported the involvement of cathepsin D in caspase 8 activation (Chiarante et al, 2018) (Figure 4). Studies performed in our laboratory with a lipophilic ZnPc also showed that ER stress induced by PDT can be propagated to the mitochondrial apoptotic pathway (Chiarante et al, 2018). In both reports, an increase of intracellular Ca 2+ concentration and higher expression levels of ER stress marker proteins, such as BIP/GRP78 and CHOP/GADD153, were demonstrated after photoactivation (Figure 5).…”

“…Later, Mfouo-Tynga et al (2014), by using ZnPcSmix, showed that the proteolytic caspase 2 cleaves and activates Bid into its truncated form (tBid) to promote mitochondrial permeabilization. Other studies with substituted ZnPcs also demonstrated that Bid cleavage occurs after irradiation (Rumie Vittar el al., 2010;Marino et al, 2013;Chiarante et al, 2018), although, in most reports, caspase 8 and lysosomal proteases have been identified as the main enzymes responsible for Bid fragmentation (Butyaert el al, 2007;Kessel and Oleinick, 2018) (Figure 4).…”

“…In particular, the first reports of ZnPcs affecting lysosomal integrity involved fluorescence microscopy studies. These works showed either a loss of the fluorescent punctuate staining of the specific LysoTracker Green probe after irradiation of different tumor cells (Yslas et al, 2007;Rumie Vittar et al, 2008;Manoto et al, 2013) or the lost of the acridine orange probe from intact acidic lysosomes (Marino et al, 2013;Sun et al, 2017;Chiarante et al, 2018). Studies performed in our laboratory either with a cationic (Marino et al, 2013) or a lipophilic (Chiarante et al, 2018) ZnPc located in lysosomes revealed that PDT treatment promotes the cytosolic release of the lysosomal protease cathepsin D in a ROS-dependent manner, since the expression levels of cathepsin D significantly diminished in the presence of antioxidants.…”

In Vivo Photodynamic Therapy With a Lipophilic Zinc(II) Phthalocyanine Inhibits Colorectal Cancer and Induces a Th1/CD8 Antitumor Immune Response

“…The reduction of Bid levels could probably be explained by the formation of the pro-apoptotic fragment tBid. In addition, in accordance with results reported in vitro [20], we found a clear down-regulation of the pro-apoptotic Bax protein. Furthermore, the lower levels of procaspase 3 suggested the activation of this pro-form by cleavage, whereas PARP-1 fragmentation indicated the inactivation of this key molecule for DNA repair [26,27].…”

“…1) [19]. Besides, we demonstrated that Pc9‐T1107 induced an apoptotic cell death both in two‐dimensional as well as three‐dimensional colon carcinoma cell cultures [19,20]. On the basis of these findings, we herein decided to explore the in vivo effect of PDT with Pc9‐T1107 in a CT26 murine colon carcinoma model.…”