Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation

In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical “molecular switch” to promote differentiation should expand the utility of this method in basic research and clinical applications

Killing Hypoxic Cell Populations in a 3D Tumor Model with EtNBS-PDT

An outstanding problem in cancer therapy is the battle against treatment-resistant disease. This is especially true for ovarian cancer, where the majority of patients eventually succumb to treatment-resistant metastatic carcinomatosis. Limited perfusion and diffusion, acidosis, and hypoxia play major roles in the development of resistance to the majority of front-line therapeutic regimens. To overcome these limitations and eliminate otherwise spared cancer cells, we utilized the cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer. Unlike standard regimens that fail to penetrate beyond ∼150 µm, EtNBS was found to not only penetrate throughout the entirety of large (>200 µm) avascular nodules, but also concentrate into the nodules' acidic and hypoxic cores. Photodynamic therapy with EtNBS was observed to be highly effective against these hypoxic regions even at low therapeutic doses, and was capable of destroying both normoxic and hypoxic regions at higher treatment levels. Imaging studies utilizing multiphoton and confocal microscopies, as well as time-lapse optical coherence tomography (TL-OCT), revealed an inside-out pattern of cell death, with apoptosis being the primary mechanism of cell killing. Critically, EtNBS-based photodynamic therapy was found to be effective against the model tumor nodules even under severe hypoxia. The inherent ability of EtNBS photodynamic therapy to impart cytotoxicity across a wide range of tumoral oxygenation levels indicates its potential to eliminate treatment-resistant cell populations

Enhancement of UVB-induced apoptosis by the chemopreventive bioflavonoid apigenin in multiple human keratinocyte models

Dissertation (Ph.D.)--University of Kansas, Pharmacology, Toxicology & Therapeutics, 2007.Topical application of the bioflavonoid apigenin (4',5,7-trihydroxyflavone) to mouse skin effectively reduces incidence and size of skin tumors caused by UVB exposure. The ability to act as a chemopreventive compound indicates that apigenin treatment alters the molecular events initiated by UVB exposure; however, the effects of apigenin treatment on UVB-irradiated keratinocytes are still not fully understood. In the present study, we aimed to study the effect of apigenin treatment and UVB exposure on human keratinocytes and to investigate how apignein may alter the biological consequences of UVB exposure. The experiments described herein, employed three models of human keratinocytes: HaCaT human keratinocyte cells, normal human keratinocytes (NHK) cultures isolated from human neonatal foreskin, and human organotypic keratinocyte cultures (OTKC). The ability of UVB to induce cycloxygenase-2 (COX-2) was investigated due to the role it is thought to play in photocarcinogenesis. The apoptotic response of keratinocytes to UVB is thought to be critical to the development of skin cancer, and therefore was investigated in multiple human keratinocyte models. Each keratinocyte model was exposed to a moderate dose of UVB (300-1000 J/m2), then treated with apigenin (0-50 μM) and harvested to assess apoptosis by Western blot analysis for poly-ADP-ribose polymerase (PARP) cleavage, annexin-V staining by flow cytometry, and/or the presence of sunburn cells. Apigenin treatment enhanced UVB-induced apoptosis more than two-fold in each of the models tested. When keratinocytes were exposed to UVB, apigenin treatment stimulated changes in Bax localization, and increased the release of cytochrome c from the mitochondria compared to UVB exposure alone. Overexpression of the anti-apoptotic protein Bcl-2 and expression of a dominant negative form of Fas-Associated Death Domain (FADDdn) led to a reduction in the ability of apigenin to enhance UVB-induced apoptosis. These results suggest enhancement of UVB-induced apoptosis by apigenin treatment involves both the stress-mediated, intrinsic pathway and the receptor-mediated, extrinsic pathway of apoptosis. The ability of apigenin to enhance UVB-induced apoptosis may explain, in part, the photochemopreventive effects of apigenin

Quantitative imaging reveals heterogeneous growth dynamics and treatment-dependent residual tumor distributions in a three-dimensional ovarian cancer model

Three-dimensional tumor models have emerged as valuable in vitro research tools, though the power of such systems as quantitative reporters of tumor growth and treatment response has not been adequately explored. We introduce an approach combining a 3-D model of disseminated ovarian cancer with high-throughput processing of image data for quantification of growth characteristics and cytotoxic response. We developed custom MATLAB routines to analyze longitudinally acquired dark-field microscopy images containing thousands of 3-D nodules. These data reveal a reproducible bimodal log-normal size distribution. Growth behavior is driven by migration and assembly, causing an exponential decay in spatial density concomitant with increasing mean size. At day 10, cultures are treated with either carboplatin or photodynamic therapy (PDT). We quantify size-dependent cytotoxic response for each treatment on a nodule by nodule basis using automated segmentation combined with ratiometric batch-processing of calcein and ethidium bromide fluorescence intensity data (indicating live and dead cells, respectively). Both treatments reduce viability, though carboplatin leaves micronodules largely structurally intact with a size distribution similar to untreated cultures. In contrast, PDT treatment disrupts micronodular structure, causing punctate regions of toxicity, shifting the distribution toward smaller sizes, and potentially increasing vulnerability to subsequent chemotherapeutic treatment

Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates

Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of “tumor-targeted, activatable photoimmunotherapy” in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease.National Science Foundation (U.S.) (R01-AR40352)National Science Foundation (U.S.) (RC1-CA146337)National Science Foundation (U.S.) (R01-CA160998)National Science Foundation (U.S.) (P01-CA084203)National Science Foundation (U.S.) (F32-CA144210

Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates

Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of “tumor-targeted, activatable photoimmunotherapy” in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease.National Science Foundation (U.S.) (R01-AR40352)National Science Foundation (U.S.) (RC1-CA146337)National Science Foundation (U.S.) (R01-CA160998)National Science Foundation (U.S.) (P01-CA084203)National Science Foundation (U.S.) (F32-CA144210