Lutetium texaphyrin: A photocatalyst that triggers pyroptosis via biomolecular photoredox catalysis

Abstract

Photon-controlled pyroptosis activation (PhotoPyro) is a promising technique for cancer immunotherapy due to its noninvasive nature, precise control, and ease of operation. Here, we report that biomolecular photoredox catalysis in cells might be an important mechanism underlying PhotoPyro. Our findings reveal that the photocatalyst lutetium texaphyrin (MLu) facilitates rapid and direct photoredox oxidation of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and various amino acids, thereby triggering pyroptosis through the caspase 3/GSDME pathway. This mechanism is distinct from the well-established role of MLu as a photodynamic therapy sensitizer in cells. Two analogs of MLu, bearing different coordinated central metal cations, were also explored as controls. The first control, gadolinium texaphyrin (MGd), is a weak photocatalyst but generates reactive oxygen species (ROS) efficiently. The second control, manganese texaphyrin (MMn), is ineffective as both a photocatalyst and a ROS generator. Neither MGd nor MMn was found to trigger pyroptosis under the conditions where MLu was active. Even in the presence of a ROS scavenger, treating MDA-MB-231 cells with MLu at concentrations as low as 50 nM still allows for pyroptosis photo-activation. The present findings highlight how biomolecular photoredox catalysis could contribute to pyroptosis activation by mechanisms largely independent of ROS.

Keywords: NAD(P)H; NIR photocatalyst; PhotoPyro; photoredox catalysis; pyroptosis.

Fig. 1.

Cell pyroptosis activation by 730 nm light-induced photoredox catalysis in cells. (A) Schematic illustration of the common photochemical MoAs involved in PhotoPyro, including photon-triggered ROS generation and photon-triggered hyperthermia; however, (B) we suggest here that biomolecular photoredox catalysis may serve as an additional MoA contributing to PhotoPyro. Figure created with Biorender.com. (C) Illustration of commonly used photocatalysts in photoredox catalysis and the key features desired for an ideal biological PC. (D) Different wavelengths of light exhibit varying tissue penetration depths. Typically, high-energy light has limited tissue penetration, whereas longer-wavelength light, particularly in the NIR range, can penetrate deeper into tissues. This makes NIR photoillumination more suitable for biomedical applications. (E) Chemical structure of the NIR PC, MLu, and its key photophysical properties.

Fig. 2.

Photoredox catalytic properties of MLu. (A) UV-vis spectral studies provide support for the photocatalytic conversion of NADH to NAD⁺ by MLu. (B) Tests of various systems as PCs for the photocatalytic production of NAD⁺. ^aYield of NAD⁺ determined after 30 s photoirradiation of 180 μM NADH in the presence of 5 μM of the indicated PC in phosphate-buffered saline (PBS) (10 mM, pH ~ 7.4) containing 10% dimethyl sulfoxide (DMSO) under air. ^bPhoton source: 730 nm LED lamp. ^cPhoton source: 660 nm LED lamp. ^dPhoton source: White LED lamp. Light power density: 35 mW/cm². ^eYield of NAD⁺ determined after 120 s 808 nm photoirradiation (100 mW/cm²) of 180 μM NADH in the presence of 5 μM of the indicated PC in PBS (10 mM, pH ~ 7.4) containing 10% DMSO under air. (C) Photocatalytic reactions with amino acids were investigated by measuring H₂O₂ generation using H₂O₂ test sticks. (D) Proposed catalytic cycle. Abbreviations: TOF, turnover frequency; TCPP, tris(1-chloro-2-propyl)phosphate; ZnPc, zinc phthalocyanine; MB, methylene blue; Met, methionine; SET, single electron transfer.

Fig. 3.

MLu-initiated cell pyroptosis activation. (A) Cellular NADH levels in MDA-MB-231 cells treated with MLu (50 nM) and subjected to light irradiation for different times (730 nm, 100 mW/cm²). (B) Change in the cellular NADH/NAD⁺ ratio seen under the indicated treatment protocols. MLu: 50 nM. Light: 730 nm, 100 mW/cm², 2 min. (C) Confocal images of Annexin-V-Alexa Fluor^TM 488-positive bubbling vesicles in MDA-MB-231 cells observed after treatment with 50 nM MLu and 730 nm photoirradiation for 2 min (100 mW/cm²). The nucleus is labeled with Hoechst 33342. (Scale bar: 10 μm.) (D) Immunoblot analysis of pyroptosis-related protein expression and corresponding statistical analysis of (E) cleaved-caspase 3 and (F) GSDME-N protein levels. (G) Release of IL-1β and (H) LDH in MDA-MB-231 cells following MLu-initiated pyroptosis activation. Light conditions for D–H: 730 nm, 100 mW/cm², 2 min. Data are presented as mean ± SD (n = 3), **P < 0.01.

Fig. 4.

Biomolecular photoredox catalysis may contribute to PhotoPyro. (A) Cell viability of MDA-MB-231 cells treated with MLu (50 nM) and subjected to photoirradiation (730 nm, 100 mW/cm², 2 min). Vitamin C (VC) was used as a ROS scavenger. (B) Strategy and control experiments were used to test the notion that biomolecular photoredox catalysis may be involved in PhotoPyro. (C) Western blot assay providing evidence for the activation of cell pyroptosis after scavenging photogenerated ROS in MDA-MB-231 cells treated with MLu (50 nM) and light (730 nm, 100 mW/cm², 2 min). (D) Chemical structures of control molecules MGd and MMn. (E) Time-dependent SYTOX green uptake of MDA-MB-231 cells after treatment with the indicated compound and subjecting to photoirradiation (730 nm, 100 mW/cm², 2 min). SYTOX green is a membrane-impenetrable probe that is normally nonfluorescent but can penetrate damaged cells to locate in the nucleus and emit in the green fluorescence channel. (F) Confocal imaging of SYTOX green in MDA-MB-231 cells. (Scale bar: 20 μm.) (G) Comparison of cell viability in MDA-MB-231 cells treated with MGd (1.5 μM) in the presence or absence of VC (0.5 mM). (H) Schematic representation of the proposed MoA of PhotoPyro involving biomolecular photoredox catalysis in cells. The figure was created using Biorender.com. Data are presented as mean ± SD (n = 3), **P < 0.01.

Fig. 5.

Anticancer properties of MLu. (A) Cytotoxicity (IC₅₀, μM) of the tested compounds (MLu, MMn, and MGd) in the MDA-MB-231, T47D, and SKBR-3 breast cancer cell lines in the absence or presence of 730 nm photoirradiation. ^aIrradiation details: 730 nm, 100 mW/cm², 2 min. ^bPhototoxicity index was calculated as the ratio of the dark IC₅₀/light IC₅₀. (B) Confocal imaging of live/dead cells in T47D 3D multicellular spheroids (MCSs) after the indicated treatments. Irradiation details: 730 nm, 100 mW/cm², 20 min. (Scale bar: 100 μm.) (C) Antiproliferative studies of MLu in T47D 3D MCSs as determined using an ATP luminescence assay kit. Data are presented as mean ± SD (n = 3), **P < 0.01.