Petras Juzenas

Mitochondrial electron transport chain (ETC) dysfunction is a major driver of bioenergetic failure, redox imbalance, and drug toxicity, yet strategies to restore oxidative phosphorylation under ETC blockade remain limited. Redox-active small molecules could, in principle, shuttle electrons from NADH to distal ETC components and oxygen, thereby modulating both respiration and reactive oxygen species (ROS) formation. Here, we show that the enzyme-independent redox cycler phenazine methosulfate (PMS) rewires mitochondrial redox circuits and restores respiration in human glioblastoma cells and cell-free systems under ETC inhibition. At subtoxic concentrations, PMS acutely increased oxygen consumption and mitochondrial superoxide generation via NADH–PMS–O₂ redox cycling, while restoring mitochondrial membrane potential and ATP synthesis under ETC blockade, and shifting metabolism away from glycolytic lactate production. This profile is consistent with a protective redox-bypass role, distinct from the pro-apoptotic effects reported following high-dose, prolonged PMS exposure. The PMS-driven restoration of electron flow, mitochondrial membrane potential, and respiratory ATP synthesis under inhibition of Complex I (rotenone), III (antimycin A and myxothiazol), and/or IV (cyanide) is consistent with direct cytochrome c reduction, as demonstrated herein, and engagement of multiple ETC redox centers, including coenzyme Q₁₀. In metformin-treated cells, PMS reversed suppression of respiration and lactate accumulation, outperforming existing redox-bypass strategies. These findings identify PMS-driven redox cycling as a previously unrecognized chemical redox-bypass mechanism that both regenerates mitochondrial bioenergetics and reshapes ROS production, suggesting a potential approach to counteract drug- and toxin-induced mitochondrial dysfunction and to exploit redox vulnerabilities in cancer.

Carbonic anhydrase IX (CAIX), overexpressed in multiple cancers but limited in normal tissue, is a promising target for radionuclide therapy. This study evaluates [²¹²Pb]Pb-MKV-509, a novel DOTA-conjugated small-molecule ligand, for CAIX-targeted alpha therapy in preclinical renal carcinoma (SK-RC-52) and colorectal (HT-29) cancer models. [²¹²Pb]Pb-MKV-509 was assessed for radiochemical purity and stability. Binding assays determined receptor density and dissociation constants. Clonogenic survival, flow cytometry (viability, DNA damage, cell cycle), and spheroid assays (cross-sectional area, doubling time) evaluated biological responses. An in vivo biodistribution study was performed in SK-RC-52 xenograft-bearing mice, with and without carbonic anhydrase pre-blocking using acetazolamide. [²¹²Pb]Pb-MKV-509 exhibited high radiochemical purity (>96%) and stability for up to 48 h. Specific binding was higher in SK-RC-52 than in HT-29 cells. Treatment induced activity-dependent clonogenic inhibition, G2/M arrest, and DNA damage, with greater sensitivity observed in SK-RC-52 cells. Clonogenic survival was reduced by 50% at 3.4 kBq/mL (SK-RC-52) and 7.1 kBq/mL (HT-29). In spheroid models, 2.5-5.0 kBq/mL delayed growth and prolonged doubling time, indicating cross-fire effects. The biodistribution study revealed significant tumor uptake (4.7%IA/g at 2 h), along with high gastrointestinal accumulation. Pretreatment with acetazolamide partially reduced uptake in the stomach and intestines as well as in the tumor. These findings highlight the potential of CAIX-targeted alpha therapy. CAIX expression and receptor density impact binding affinity and therapeutic response. The study demonstrates the importance of 3D tumor models in evaluating alpha-particle cross-fire effects. Further ligand optimization is warranted to enhance tumor specificity and minimize off-target uptake.

The alpha-emitting radioligand [²¹²Pb]Pb-AB001, targeting prostate-specific membrane antigen (PSMA), is a promising therapy approach for prostate cancer. Bromodomain and extra-terminal (BET) protein inhibitors, such as AZD5153 and JQ1, disrupt oncogenic transcriptional programs by altering chromatin structure. This study evaluated whether BET inhibition enhances the efficacy of radioligand therapy. Cytotoxic effects of [²¹²Pb]Pb-AB001 alone and in combination with BET inhibitors were assessed in 2D monolayers and a 3D spheroid model of PSMA-positive C4-2 prostate cancer cells. Cell viability, cell cycle alterations, and DNA damage were assessed using viability assays and flow cytometry. Spheroid growth and viability were assessed by fluorescence microscopy. AZD5153 was more potent than JQ1 in reducing cell proliferation. [²¹²Pb]Pb-AB001 induced activity- and time-dependent cytotoxicity with a delayed apoptotic response. BET inhibitors induced G1 arrest, while [²¹²Pb]Pb-AB001 caused G2 arrest. Combination treatment reduced cell viability in an additive manner but did not further affect cell cycle distribution or increase apoptosis compared with [²¹²Pb]Pb-AB001 alone. γH2AX staining in 2D models showed an activity- and time-dependent increase in DNA damage at 1, 3 and 6 days post-treatment with [²¹²Pb]Pb-AB001. BET inhibitors alone induced minimal γH2AX, and combination treatments did not enhance DNA damage beyond [²¹²Pb]Pb-AB001 alone. In 3D spheroids, combination treatment led to synergistic growth suppression. In conclusion, these findings indicate that therapeutic inhibition of BET bromodomain in combination with the alpha-emitting radioligand [²¹²Pb]Pb-AB001 could significantly enhance tumour control and should be further evaluated in metastatic prostate cancer models.

Background/Objectives: With the increasing prevalence of Crohn’s disease (CD), treatment options for patients who fail conventional and advanced therapy are highly needed. Therefore, we explored the safety and efficacy of extracorporeal photopheresis (ECP) using 5-aminolevulinic acid (ALA) and blue light (405 nm). Methods: Patients with active CD who failed or were intolerant to biological therapy were eligible. Mononuclear cells (90 mL) were collected from each patient using a Spectra Optia^® apheresis system and diluted with 100 mL of 0.9% sodium chloride in a collection bag. The cells were incubated with ALA at a concentration of 3 millimolar (mM) for 60 min ex vivo and illumination with an LED blue light (405 nm) source (BLUE-PIT^®) before reinfusion to the patient. Recording of vital signs and adverse events were regularly performed. At week 13, we assessed the patients with colonoscopy, the Harvey Bradshaw Index (HBI), the Inflammatory Bowel disease Health Related Quality of Life Questionnaire, and the measurement of serum C-reactive protein and fecal calprotectin (FC) levels. Biopsies of the intestines were taken for immunohistochemistry. Results: Seven patients were included. Four patients completed the treatments, with a total of 24 treatments. Three of the four patients achieved a favorable response, including a lower HBI, lower FC levels, and/or endoscopic improvement. No significant adverse events were observed. The remaining three patients received only one, three, or five treatments due to technical difficulties, medical reasons, or the withdrawal of informed consent. Conclusions: ALA-based ECP appears safe and seems to give some clinical improvement for the patients with active CD who failed to respond to conventional and advanced therapies.

Background/Objectives: With the increasing prevalence of Crohn’s disease (CD), treatment op-tions for patients who fail conventional and advanced therapy are highly needed. Therefore, we explored the safety and efficacy of extracorporeal photopheresis (ECP) using 5-aminolevulinic acid (ALA) and blue light (405 nm). Methods: Patients with active CD who failed or were intolerant to biological therapy were eligi-ble. Mononuclear cells (90 mL) were collected from each patient using a Spectra Optia® apheresis system and diluted with 100 mL of 0.9% sodium chloride in a collection bag. The cells were in-cubated with ALA at a concentration of 3 millimolar (mM) for 60 minutes ex vivo and illumina-tion with a LED blue-light (405 nm) source (BLUE-PIT®) before reinfusion to the patient. Vital signs and adverse events were regularly performed. At week 13 we assessed the patients with colonoscopy, the Harvey Bradshaw Index (HBI), the Inflammatory Bowel disease Health Related Quality of Life Questionnaire, and measurement of serum C-reactive protein, and fecal calprotec-tin (FC) levels. Biopsies of the intestines were taken for immunohistochemistry. Results: Seven patients were included. Four patients completed, with in total 24 treatments. Three of the four achieved a response, including lower HBI, FC and/or endoscopic improvement. No significant adverse events were observed. The remaining three patients received only one, three or five treatments due to technical difficulties, medical reasons, or the withdrawal of informed con-sent. Conclusion: ALA-based ECP appears safe and seem to give some clinical improvement for the patients with active CD who failed to respond to conventional and advanced therapies.

Extracorporeal photopheresis (ECP) is a therapeutic modality used for T-cell-mediated disorders. This approach involves exposing isolated white blood cells to photoactivatable 8-methoxypsoralen (8-MOP) and UVA light, aiming to induce apoptosis in T-cells and thereby modulate immune responses. However, conventional 8-MOP-ECP lacks cell selectivity, killing both healthy and diseased cells, and has shown limited treatment efficacy. An alternative approach under investigation involves the use of 5-aminolevulinic acid (ALA) in conjunction with light, referred to as ALA-based photodynamic therapy. Our previous ex vivo studies suggest that ALA-ECP exhibits greater selectivity and efficiency in killing T-cells derived from patients with T-cell-mediated disorders compared to those treated with 8-MOP-ECP. We have conducted a clinical phase I–(II) study evaluating ALA-ECP safety and tolerability in cutaneous T-cell lymphoma (CTCL). Here, 20 ALA-ECP treatments were administered to one CTCL patient, revealing no significant changes in vital signs. Two adverse events were reported; both evaluated by the Internal Safety Review Committee as non-serious. In addition, five conceivable events with mainly mild symptoms took place. During the study period, a 53% reduction in skin involvement and a 50% reduction in pruritus was observed. In conclusion, the results indicate that ALA-ECP treatment is safe and well tolerated.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) which is the precursor of the photosensitizer protoporphyrin IX (PpIX) is an available treatment for several diseases. ALA-PDT induces the apoptosis and necrosis of target lesions. We have recently reported the effects of ALA-PDT on cytokines and exosomes of human healthy peripheral blood mononuclear cells (PBMCs). This study has investigated the ALA-PDT-mediated effects on PBMC subsets from patients with active Crohn’s disease (CD). No effects on lymphocyte survival after ALA-PDT were observed, although the survival of CD3⁻/CD19⁺ B-cells seemed slightly reduced in some samples. Interestingly, ALA-PDT clearly killed monocytes. The subcellular levels of cytokines and exosomes associated with inflammation were widely downregulated, which is consistent with our previous findings in PBMCs from healthy human subjects. These results suggest that ALA-PDT may be a potential treatment candidate for CD and other immune-mediated diseases.

Photodynamic therapy (PDT) is a non-invasive therapeutic modality based on the interaction between a photosensitive molecule called photosensitizer (PS) and visible light irradiation in the presence of oxygen molecule. Protoporphyrin IX (PpIX), an efficient and widely used PS, is hampered in clinical PDT by its poor water-solubility and tendency to self-aggregate. These features are strongly related to the PS hydrophilic–lipophilic balance. In order to improve the chemical properties of PpIX, a series of amphiphilic PpIX derivatives endowed with PEG₅₅₀ headgroups and hydrogenated or fluorinated tails was synthetized. Hydrophilic–lipophilic balance (HLB) and log p-values were computed for all of the prepared compounds. Their photochemical properties (spectroscopic characterization, photobleaching, and singlet oxygen quantum yield) were also evaluated followed by the in vitro studies of their cellular uptake, subcellular localization, and photocytotoxicity on three tumor cell lines (4T1, scc-U8, and WiDr cell lines). The results confirm the therapeutic potency of these new PpIX derivatives. Indeed, while all of the derivatives were perfectly water soluble, some of them exhibited an improved photodynamic effect compared to the parent PpIX.

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), a precursor to the potent photosensitizer, protoporphyrin IX (PpIX), is an established modality for several malignant and premalignant diseases. This treatment is based on the light-activated PpIX in targeted lesions. Although numerous studies have confirmed the necrosis and apoptosis involved in the mechanism of action of this modality, little information is available for the change of exosome levels after treatment. We report from the first study on the effects of ALA-PDT on cytokines and exosomes of human healthy peripheral blood mononuclear cells (PBMCs). The treatment reduced the cytokines and exosomes studied, although there was variation among individual PBMC samples. This reduction is consistent with PDT-mediated survivals of subsets of PBMCs. More specifically, the ALA-PDT treatment apparently decreased all pro-inflammatory cytokines included, suggesting that this treatment may provide a strong anti-inflammatory effect. In addition, the treatment has decreased the levels of different types of exosomes, the HLA-DRDPDQ exosome in particular, which plays an important role in the rejection of organ transplantation as well as autoimmune diseases. These results may suggest future therapeutic strategies of ALA-PDT.

The authors wish to make the following corrections to this paper