Background and Objective We investigated the relationship among heat shock protein 70 (hsp70) promoter activation, extracellular HSP70 protein levels, and tumor cure in an animal model of meso-tetrahydroxyphenyl chlorin (mTHPC; Foscan®)-mediated photodynamic therapy (PDT). Materials and Methods Using Western blot analysis, we compared HSP70 protein levels in control and PDT-treated EMT6 cells with the amplitude of hsp70-promoter driven green fluorescent protein (GFP) expression in identically treated, stably transfected hsp70-GFP/EMT6 cells. A clonogenic survival assay was performed to assess the relationship among promoter activation, HSP70 levels, and cell survival in vitro. Tumor growth studies with this transfected cell line were performed to examine responses to fluences from 0.1 to 10 J cm⁻², which ranged from sub-curative to curative. In vivo stereofluorescence and confocal fluorescence imaging were used to assess the temporal kinetics in hsp70 activation in tumors subjected to these fluences and the intratumor spatial correlation between hsp70 induction and extracellular levels of HSP70, respectively. Results Maximum GFP expression and HSP protein levels in cells were observed at PDT doses that corresponded to 30% cell survival. The relative changes in GFP and HSP70 protein accumulation as analyzed using Western immunoblots agreed very well, thereby confirming the validity of fluorescent reporter assessment of gene expression in our studies. In vivo imaging revealed that hsp70 promoter-driven GFP expression and accumulation of extracellular HSP70 in PDT-treated tumors subjected to non-curative doses exhibit minimal spatial correlation. There is a strong correlation between mTHPC-PDT doses that result in long-term tumor cure and those that cause high levels of surface exposed or extracellularly released HSP70s. Conclusion Treatment conditions that induce strong promoter activation do not correspond to tumor cure. PDT doses that result in long-term tumor growth control also produce significant accumulation of extracellular HSP70. Lasers Surg. Med. 43:632–643, 2011.

Meso-tetra-hydroxyphenyl-chlorin (mTHPC, Foscan), a promising photosensitizer for photodynamic therapy (PDT), is approved in Europe for the palliative treatment of head and neck cancer. Based on work in mice that investigated optimal tumor accumulation, clinical protocols with Foscan typically employ an interval of 96 h between systemic sensitizer administration and irradiation. However, recent studies in mouse tumor models have demonstrated significantly improved long-term tumor response when irradiation is performed at shorter drug-light intervals of 3 and 6 h. Using a previously published theoretical model of microscopic PDT dosimetry and informed by experimentally determined photophysical properties and intratumor sensitizer concentrations and distributions, we calculated photodynamic dose depositions following mTHPC-PDT for drug-light intervals of 3, 6, 24, and 96 h. Our results demonstrate that the singlet oxygen dose to the tumor volume does not track even qualitatively with tumor responses for these four drug-light intervals. Further, microscopic analysis of simulated singlet oxygen deposition shows that in no case do any subpopulations of tumor cells receive a threshold dose. Indeed, under the conditions of these simulations more than 90% of the tumor volume receives a dose that is approximately 20-fold lower than the threshold dose for mTHPC. Thus, in this evaluation of mTHPC-PDT at various drug-light intervals, any PDT dose metric that is proportional to singlet oxygen creation and/or deposition would fail to predict the tumor response. In situations like this one, other reporters of biological response to therapy would be necessary.

Light scattering measurements made at visible wavelengths have the ability to quantify subcellular morphology. Apoptosis, or programmed cell death, is associated with distinct morphological signatures such as mitochondrial swelling and nuclear condensation as well as characteristic biochemical signaling pathways, many of which are initiated by the release of cytochrome c from the mitochondria into the cytosol. In this Letter, we examine the time course of mitochondrial morphology changes as reported by light scattering and the subcellular location of cytochrome c measured by immunofluorescence microscopy in response to intracellular cell death signaling induced by photodynamic damage to lysosomes. We report that within this system, release of cytochrome c from the mitochondria into the cytosol occurs approximately simultaneously with mitochondrial-morphology-induced light scattering changes, providing further evidence that light scattering has the potential to play an important role in future studies of cell death biology.

In view of the ethno medicinal use of Enhydra fluctuans for the treatment of kidney stones; the present study aimed to elucidate the molecular mechanisms involved in the amelioration of nephrolithiasis through a network pharmacology approach. The phytoconstituents were queried in DIGEP-Pred to identify the regulated proteins. The modulated proteins were then enriched in the STRING to predict the protein-protein interactions and the probably regulated pathways were traced in the Kyoto Encyclopedia of Genes and Genomes. Further, the network was constructed using Cytoscape ver 3.5.1. Results showed that β-carotene was found to be regulating maximum targets i.e. 26. In addition, 63 proteins were triggered by the components in which the vitamin D receptor was targeted by the maximum phytoconstituents i.e. 16. The enrichment analysis identified the regulation of 67 pathways in which fluid shear stress and atherosclerosis-associated pathways (KEGG entry hsa05418) regulated ten genes. Further, protein kinase C-α was traced in 23 different pathways. In addition, the majority of the regulated genes were identified from the extracellular space via the modulation of 43 genes. Also, nuclear receptor activity had the maximum molecular function via the regulation of 7 genes. Likewise, the response to organic substance was predicted to trigger the top genes i.e. 43. In contrast, Stigmasterol, Baicalein-7-o-glucoside, and Kauran-16-ol were found to have a high affinity to bind with the VDR receptor confirmed by the molecular modelling and the dynamics. Hence, the study elucidated the probable molecular mechanisms of E. fluctuans in managing nephrolithiasis and identified the lead molecules, their targets, and possible pathways. Communicated by Ramaswamy H. Sarma

Meso-tetra-hydroxyphenyl-chlorin (mTHPC) is one of the most potent photosensitizers currently available for clinical photodynamic therapy (PDT). However the reason or reasons for its high photodynamic efficacy remain(s) unresolved. To investigate the PDT efficacy of mTHPC vs Photofrin we use the knowledge of photophysical parameters extracted from the analysis of oxygen electrode measurements in spheroids to compute and compare their respective singlet oxygen (1O2) dose depositions. The electrode measurements indirectly report the bleaching kinetics of mTHPC and indicate that its photobleaching mechanism is consistent with 1O2-mediated reactions. mTHPC's photodegradation via 1O2 reactions is confirmed by a more direct evaluation of the spatially resolved fluorescence in confocal sections of intact spheroids during irradiation. The PDT efficacy comparisons establish that mTHPC's enhanced potency may be accounted for completely on the basis of its ability to sequester tightly in cells and its photophysical properties, in particular its higher extinction coefficient at a redshifted wavelength. We extend the efficacy comparison to include the influence of hemoglobin absorption of PDT treatment light and show that incorporating the influence of wavelength-dependent light attenuation in tissue further contributes to significantly higher efficacy for mTHPC- vs Photofrin-PDT.

Human soluble epoxide hydrolase (sEH), a dual-functioning homodimeric enzyme with hydrolase and phosphatase activities, is known for its pivotal role in the hydrolysis of epoxyeicosatrienoic acids. Inhibitors targeting sEH have shown promising potential in the treatment of various life-threatening diseases. In this study, we employed a range of in silico modeling approaches to investigate a diverse dataset of structurally distinct sEH inhibitors. Our primary aim was to develop predictive and validated models while gaining insights into the structural requirements necessary for achieving higher inhibitory potential. To accomplish this, we initially calculated molecular descriptors using nine different descriptor-calculating tools, coupled with stochastic and non-stochastic feature selection strategies, to identify the most statistically significant linear 2D-QSAR model. The resulting model highlighted the critical roles played by topological characteristics, 2D pharmacophore features, and specific physicochemical properties in enhancing inhibitory potential. In addition to conventional 2D-QSAR modeling, we implemented the Transformer-CNN methodology to develop QSAR models, enabling us to obtain structural interpretations based on the Layer-wise Relevance Propagation (LRP) algorithm. Moreover, a comprehensive 3D-QSAR analysis provided additional insights into the structural requirements of these compounds as potent sEH inhibitors. To validate the findings from the QSAR modeling studies, we performed molecular dynamics (MD) simulations using selected compounds from the dataset. The simulation results offered crucial insights into receptor–ligand interactions, supporting the predictions obtained from the QSAR models. Collectively, our work serves as an essential guideline for the rational design of novel sEH inhibitors with enhanced therapeutic potential. Importantly, all the in silico studies were performed using open-access tools to ensure reproducibility and accessibility.