Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.     

The Course of Immune Stimulation by Photodynamic Therapy: Bridging Fundamentals of Photochemically Induced Immunogenic Cell Death to the Enrichment of T‐Cell Repertoire

Photodynamic therapy (PDT) is a potentially immunogenic and FDA‐approved antitumor treatment modality that utilizes the spatiotemporal combination of a photosensitizer, light and oftentimes oxygen, to generate therapeutic cytotoxic molecules. Certain photosensitizers under specific conditions, including ones in clinical practice, have been shown to elicit an immune response following photoillumination. When localized within tumor tissue, photogenerated cytotoxic molecules can lead to immunogenic cell death (ICD) of tumor cells, which release damage‐associated molecular patterns and tumor‐specific antigens. Subsequently, the T‐lymphocyte (T cell)–mediated adaptive immune system can become activated. Activated T cells then disseminate into systemic circulation and can eliminate primary and metastatic tumors. In this review, we will detail the multistage cascade of events following PDT of solid tumors that ultimately lead to the activation of an antitumor immune response. More specifically, we connect the fundamentals of photochemically induced ICD with a proposition on potential mechanisms for PDT enhancement of the adaptive antitumor response. We postulate a hypothesis that during the course of the immune stimulation process, PDT also enriches the T‐cell repertoire with tumor‐reactive activated T cells, diversifying their tumor‐specific targets and eliciting a more expansive and rigorous antitumor response. The implications of such a process are likely to impact the outcomes of rational combinations with immune checkpoint blockade, warranting investigations into T‐cell diversity as a previously understudied and potentially transformative paradigm in antitumor photodynamic immunotherapy.     

Complement activation cascade and its regulation: relevance for the response of solid tumors to photodynamic therapy

The complement system has emerged as a prominent participant in host response elicited following treatment of solid tumors by photodynamic therapy (PDT). Activity of the complement system is tightly controlled by a series of endogenous regulatory proteins. The expression of decay-accelerating factor (DAF), complement-receptor-1-related protein y (Crry), and protectin, which are the three major mouse membrane-bound complement regulatory proteins (mCRPs), was examined following treatment of murine squamous cell carcinomas SCCVII by PDT mediated by the photosensitizer Photofrin. A marked decrease was detected in the expression of all three mCRPs on cancer cells from tumors following PDT, indicating that these cells were made more vulnerable to complement attack. In order to amplify this effect, following PDT mice were injected with antibodies neutralizing either Crry, protectin, or DAF. With anti-Crry and anti-protectin this resulted in increased tumor cure rate compared to PDT alone, while the contrary was observed with PDT plus anti-DAF combination (presumably owing to additional role of DAF in T cell signaling). Further examination including other complement regulatory proteins showed that combining antitumor PDT with antibody-mediated neutralization of factor H (soluble negative complement regulator) or injection of properdin (positive complement regulator) increased the cure rates of PDT-treated tumors. The use of various agents promoting complement activity appears promising for employment as adjuvants to PDT.     

NUCLEAR FACTOR κB BINDING ACTIVITY IN MOUSE L1210 CELLS FOLLOWING PHOTOFRIN II-MEDIATED PHOTOSENSITIZATION

Clinical photodynamic therapy (PDT) uses the photosensitizer photofnn II to produce singlet molecular oxygen and other reactive oxygen intermediates for localized tumor tissue cytotoxicity. In this report, we show that PDT enhances the DNA binding activity of nuclear factor kappa B (NFkB), a transacti vator of cytokine gene expression. Photosensitization following a 16 h incubation of photofrin II induced NFkB binding activity in mouse leukemia L1210 cells 10-fold above that observed in exponentially growing cultures. Serum starvation, as well as drug-alone and light-alone controls, elevated basal NF k B binding activity two- to three-fold. Upstream stimulatory factor binding activity was not modulated by any of the cell treatments and was used to standardize gel mobility shift data. This study identifies porphynn-mediated PDT as an inducer of NF k B binding activity, extending recent findings that NF k B activation is a general response to oxidative stress.     

Epigenetically Enhanced Photodynamic Therapy (ePDT) is Superior to Conventional Photodynamic Therapy for Inducing Apoptosis in Cutaneous T‐Cell Lymphoma

Conventional photodynamic therapy with aminolevulinate (ALA‐PDT) selectively induces apoptosis in diseased cells and is highly effective for treating actinic keratoses. However, similar results are achieved only in a subset of patients with cutaneous T‐cell lymphoma (CTCL). Our previous work shows that the apoptotic resistance of CTCL correlates with low expression of death receptors like Fas cell surface death receptor (FAS), and that methotrexate upregulates FAS by inhibiting the methylation of its promoter, acting as an epigenetic derepressor that restores the susceptibility of FAS‐low CTCL to caspase‐8‐mediated apoptosis. Here, we demonstrate that methotrexate increases the response of CTCL to ALA‐PDT, a concept we refer to as epigenetically enhanced PDT (ePDT). Multiple CTCL cell lines were subjected to conventional PDT versus ePDT. Apoptotic biomarkers were analyzed in situ with multispectral imaging analysis of immunostained cells, a method that is quantitative and 5× more sensitive than standard immunohistology for antigen detection. Compared to conventional PDT or methotrexate alone, ePDT led to significantly greater cell death in all CTCL cell lines tested by inducing greater activation of caspase‐8‐mediated extrinsic apoptosis. Upregulation of FAS and/or tumor necrosis factor‐related apoptosis‐inducing ligand pathway components was observed in different CTCL cell lines. These findings provide a rationale for clinical trials of ePDT for CTCL.     

Angiogenesis induced by photodynamic therapy in normal rat brains

Angiogenesis promotes tumor growth and invasiveness in brain. Because brain injury often induces expression of angiogenic-promoting molecules, we hypothesize that oxidative insult induced by photodynamic therapy (PDT) could lead to an endogenous angiogenic response, possibly diminishing the efficacy of PDT treatment of tumors. Therefore, we sought to establish whether PDT induced an angiogenic response within the nontumored brain. PDT using Photofrin as a sensitizer at an optical dose of 140 J/cm2 was performed on normal rat brain (n = 30). Animals were sacrificed at 24 h, and 1, 2, 3 and 6 weeks after PDT treatment. Fluorescein isothiocyanatedextran perfusion was performed, and brains were fixed for immunohistological study. Immunostaining revealed that vascular endothelial growth factor (VEGF) expression increased within the PDT-treated hemisphere 1 week after treatment and remained elevated for 6 weeks. Three-dimensional morphologic analysis of vasculature within PDT-treated and contralateral brain demonstrated PDT-induced angiogenesis, as indicated by a significant increase in vessel connectivity (P < 0.001) concomitant with decreased (P < 0.05) mean segment length compared with vessels within the contralateral hemisphere. Volumetric measurement of angiogenic regions indicate that neovascular expansion continued for 4 weeks after PDT. These data demonstrate that PDT induces VEGF expression and neovascularization within normal brain. Because angiogenesis promotes growth and invasiveness of tumor, antagonizing this endogenous angiogenic response to PDT may present a practical means to enhance the efficacy of PDT.     

Nitric Oxide-Mediated Resistance to Antitumor Photodynamic Therapy

As an antitumor modality based on sensitizer photoexcitation by tumor-directed light, photodynamic therapy (PDT) has the advantage of being site-specific compared with conventional chemotherapy or radiotherapy. Like these other therapies, however, PDT is often limited by pre-existing or acquired resistance. One type of resistance, discovered in the author’s laboratory, involves nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) in tumor cells. Using human breast, prostate and brain cancer cell lines, we have shown that iNOS is dramatically upregulated after a moderate PDT challenge sensitized by 5-aminolevulinic acid-induced protoporphyrin IX. The elevated NO not only elicited a greater resistance to cell photokilling, but also an increase in the growth and migration/invasion rate of surviving cells. Greater iNOS/NO-based resistance was also demonstrated at the in vivo level using a breast tumor xenograft model. More recent studies have shown that NO from PDT-targeted cells can stimulate a progrowth/promigration response in non-targeted bystander cells. These novel effects of NO, their negative impact on PDT efficacy and possible mitigation thereof by anti-iNOS/NO pharmacologic agents will be discussed.     

Modeling Epidermal Growth Factor Inhibitor‐mediated Enhancement of Photodynamic Therapy Efficacy Using 3D Mesothelioma Cell Culture

We have demonstrated that lung‐sparing surgery with intraoperative photodynamic therapy (PDT) achieves remarkably extended survival for patients with malignant pleural mesothelioma (MPM). Nevertheless, most patients treated using this approach experience local recurrence, so it is essential to identify ways to enhance tumor response. We previously reported that PDT transiently activates EGFR/STAT3 in lung and ovarian cancer cells and inhibiting EGFR via erlotinib can increase PDT sensitivity. Additionally, we have seen higher EGFR expression associating with worse outcomes after Photofrin‐mediated PDT for MPM, and the extensive desmoplastic reaction associated with MPM influences tumor phenotype and therapeutic response. Since extracellular matrix (ECM) proteins accrued during stroma development can alter EGF signaling within tumors, we have characterized novel 3D models of MPM to determine their response to erlotinib combined with Photofrin‐PDT. Our MPM cell lines formed a range of acinar phenotypes when grown on ECM gels, recapitulating the locally invasive phenotype of MPM in pleura and endothoracic fascia. Using these models, we confirmed that EGFR inhibition increases PDT cytotoxicity. Together with emerging evidence that EGFR inhibition may improve survival of lung cancer patients through immunologic and direct cell killing mechanisms, these results suggest erlotinib‐enhanced PDT may significantly improve outcomes for MPM patients.     

In vivo resistance to photofrin-mediated photodynamic therapy in radiation-induced fibrosarcoma cells resistant to in vitro Photofrin-mediated photodynamic therapy.

The effects of Photofrin-mediated photodynamic therapy (PDT) on the in vitro cell survival and in vivo tumor growth of murine radiation-induced fibrosarcoma (RIF) cell tumors have been examined following in vivo PDT treatment of tumors. The response to in vivo PDT is examined in tumors derived from RIF-1 mouse fibrosarcoma cells and in tumors derived from RIF-8A cells, which show in vitro resistance to PDT. A significant reduction in tumor volume is observed over the first three days following in vivo PDT treatment of either 5 or 10 mg/ kg. The reduction in tumor volume is greater for a 10 compared to a 5 mg/ml dose and occurs to a similar extent for both RIF-1 and RIF-8A tumors. The re-growth is significantly delayed for RIF-1 compared to RIF-8A tumors, indicating a greater response for RIF-1 tumors compared to RIF-8A tumors following PDT. A reduced response of the RIF-8A compared to the RIF-1 tumor cells is also observed in the clonogenic survival of cells from tumors that were excised and explanted in vitro immediately following in vivo PDT treatment. These data indicate that the intrinsic cell sensitivity to PDT is an important component in the mechanism that leads to tumor response following in vivo photodynamic therapy.     

STRESS PROTEIN EXPRESSION IN MURINE TUMOR CELLS FOLLOWING PHOTODYNAMIC THERAPY WITH BENZOPORPHYRIN DERIVATIVE

Abstract— Photodynamic therapy (PDT) has been proven as a method of tumor eradication and is currently being used clinically to treat a wide variety of malignancies. Although it is understood that the interaction of light and sensitizer results in the production of potentially damaging oxygen species, the mechanism by which tumors are destroyed has yet to be defined fully. Using a new porphyrin sensitizer, benzoporphyrin derivative(BPD), we examined protein expression in murine tumor cells following treatment as an indication of molecular changes to target tissue concurrent with PDT-mediated damage. In order to assess the relevance of the results obtained using an in vitro PDT model, metabolic labeling of proteins synthesized subsequent to PDT was performed both in tumor cells grown and treated in tissue culture dishes and in cells explanted from PDT-treated solid tumors. We observed that the oxidative stress associated with PDT-resulted in the induction of a number or proteins corresponding to a set of heat-shock or stress proteins, and that the pattern of expression was similar when tumor cells were treated in vitro and in vivo . These results support the use of in vitro models in the dissection of the molecular erects of PDT and provide the foundation for future experiments that will examine the role of the immune system in tumor eradication by PDT.     

Combination therapy with antiangiogenic treatment and photodynamic therapy for the nude mouse bearing U87 glioblastoma

The objective of this study was to evaluate the effects of combination therapy with photodynamic therapy (PDT) and a novel antiangiogenic regimen using monoclonal antibodies against both vascular endothelial growth factor receptors (VEGFR)-1 (MF1) and VEGFR-2 (DC101) on intracranial glioblastoma xenografts in nude mice. Nude mice bearing intracerebral U87 glioblastoma were treated with PDT and the antiangiogenic regimen (MF1 and DC101) either alone or in combination, while those left untreated served as tumor controls. Tumor volume and animal survival time were analyzed to evaluate the outcome of different treatment modalities. In addition, the immunohistochemical expression of VEGF in the brain adjacent to the tumor, von Willebrand factor (vWF), apoptotic, and proliferative markers in the tumor area were examined. PDT or MF1 + DC101 alone significantly reduced the tumor volume and prolonged the survival time of glioma-implanted animals. Combined therapy markedly reduced tumor volume and increased survival time with significantly better outcomes than both monotherapies. Both vWF and VEGF levels significantly increased after PDT while they both significantly decreased after antiangiogenic treatment, compared with no treatment. PDT plus antiangiogenic treatment led to significant decreases in both vWF and VEGF expression, compared with PDT alone. Either PDT or antiangiogenic treatment alone significantly increased tumor cell apoptosis compared with no treatment, while combination therapy resulted in further augmentation of apoptosis. Antiangiogenic treatment with or without PDT significantly decreased tumor cell proliferation, compared with either no treatment or PDT alone. In summary, we demonstrate both significant inhibition of tumor growth and extended survival of mice treated by the combination therapy with PDT and antiangiogenic agents, compared with each single treatment, suggesting that the combination therapy may be a promising strategy to improve clinical outcomes in glioblastoma.     

Combination of phosphatidylinositol 3-kinases pathway inhibitor and photodynamic therapy in endothelial and tumor cells

Tumor recurrence due to incomplete eradication of tumor cells is a major problem facing current cancer therapies. To overcome this problem, it is necessary to enhance cell killing and/or prevent cell regrowth after treatment. Because phosphatidylinositol 3-kinases (PI3K) pathway plays an important role in stimulating cell survival and growth, we studied the feasibility of using a PI3K pathway inhibitor NVP-BEZ235 (BEZ235) to enhance the effectiveness of vascular-targeted photodynamic therapy (vPDT) with verteporfin. We found that BEZ235 or PDT alone significantly inhibited cell growth in both SVEC endothelial and PC-3 prostate cancer cells, although SVEC cells appeared to be more responsive than PC-3 cells. Autophagy was detected after both BEZ235 and verteporfin-PDT in both cell lines. Autophagy appeared to protect cells from PDT-induced cell death because inhibition of autophagy increased cell death. Autophagic flux assay revealed that PDT actually decreased autophagic flux especially at a high dose of verteporfin. Combination of BEZ235 and PDT caused greater inhibition of PI3K signaling pathway, leading to enhanced cell growth inhibition in both cell lines. SVEC cells exhibited a higher sensitivity towards such a combination than PC-3 cells. Our data indicated that BEZ235 in combination with PDT provides a promising approach of enhancing therapeutic response.     

Bioluminescence imaging of the response of rat gliosarcoma to ALA-PpIX-mediated photodynamic therapy

Photodynamic therapy (PDT) is a promising modality for the treatment of solid tumors that combines a photosensitizing agent and light to produce cytotoxic reactive oxygen species that lead to tumor cell death. The recent introduction of bioluminescence imaging (BLI), involving the use of the luciferase gene (luc) transferred into target tumor cells, followed by systemic administration of luciferin and detection of the emitted visible chemiluminescence photons, offers the potential for longitudinal imaging of tumor growth and therapeutic response in single animals. We demonstrate in this study the first results of the use of BLI to assess the response of an intracranial brain tumor model (9L rat gliosarcoma) to aminolevulinic acid (ALA)-mediated PDT. Complementary in vitro experiments with the luciferase-transfected 9L cells show that the decrease in the luminescent signal after PDT correlates with cell kill. In vivo imaging shows a decrease in the BLI signal from the tumor after ALA-PDT treatment, followed by tumor regrowth. Furthermore, preliminary studies using cells transfected with a hypoxia-responsive vector show an increase in bioluminescence within 4 h after Photofrin-mediated PDT, demonstrating the ability to observe stress-gene responses. These results suggest that BLI can be used to provide spatiotemporal information of intracranial brain tumor responses after PDT and may serve as a valuable response-endpoint measure.