Decreased phototoxicity of photodynamic therapy by Cx32/Cx26-composed GJIC: A "Good Samaritan" effect.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) has been widely used to treat malignant tumors. Our previous studies indicated that connexin (Cx) 32- and Cx26-composed gap junctional intercellular communication (GJIC) could improve the phototoxicity of PDT. However, the role of heterotypic Cx32/Cx26-formed GJIC in PDT phototoxicity is still unknown. Thus, the present study was aimed to investigate the effect of Cx32/Cx26-formed GJIC on PDT efficacy. METHODS: CCK8 assay was used to detect cell survival after PDT. Western blot assay was utilized to detect Cx32/Cx26 expression. "Parachute" dye-coupling assay was performed to measure the function of GJ channels. The intracellular Ca2+ concentrations were determined using flow cytometer. ELISA assay was performed to detect the intracellular levels of PGE2 and cAMP. RESULTS: The present study demonstrates there is a Cx32/Cx26-formed GJIC-dependent reduction of phototoxicity when cells were exposure to low concentration of Photofrin. Such a protective action is missing at low cell density due to the lack of GJ coupling. Under high-cell density condition, where there is opportunity for the cells to contact each other and form GJ, suppressing Cx32/Cx26-formed GJIC by either inhibiting the expression of Cx32/Cx26 or pretreating with GJ channel inhibitor augments PDT phototoxicity after cells were treated with at 2.5 µg/ml Photofrin. The above results suggest that at low Photofrin concentration, the presence of Cx32/Cx26-formed GJIC may decrease the phototoxicity of PDT, leading to the insensitivity of malignant cells to PDT treatment. The GJIC-mediated PDT insensitivity was associated with Ca2+ and prostaglandin E2 (PGE2 ) signaling pathways. CONCLUSION: The present study provides a cautionary note that for tumors expressing Cx32/Cx26, the presence of Cx32/Cx26-composed GJIC may cause the resistance of tumor cells to PDT. Oppositely, treatment strategies designed to downregulate the expression of Cx32/Cx26 or restrain the function of Cx32/Cx26-mediated GJIC may increase the sensitivity of malignant cell to PDT. Lasers Surg. Med. 51:301-308, 2019. © 2019 Wiley Periodicals, Inc.

Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy.

BACKGROUND: Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. METHODS: In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. RESULTS: Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. CONCLUSIONS: Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.

Development of photodynamic therapy regimens that control primary tumor growth and inhibit secondary disease.

Effective therapy for advanced cancer often requires treatment of both primary tumors and systemic disease that may not be apparent at initial diagnosis. Numerous studies have shown that stimulation of the host immune system can result in the generation of anti-tumor immune responses capable of controlling metastatic tumor growth. Thus, there is interest in the development of combination therapies that both control primary tumor growth and stimulate anti-tumor immunity for control of metastatic disease and subsequent tumor growth. Photodynamic therapy (PDT) is an FDA-approved anticancer modality that has been shown to enhance anti-tumor immunity. Augmentation of anti-tumor immunity by PDT is regimen dependent, and PDT regimens that enhance anti-tumor immunity have been defined. Unfortunately, these regimens have limited ability to control primary tumor growth. Therefore, a two-step combination therapy was devised in which a tumor-controlling PDT regimen was combined with an immune-enhancing PDT regimen. To determine whether the two-step combination therapy enhanced anti-tumor immunity, resistance to subsequent tumor challenge and T cell activation and function was measured. The ability to control distant disease was also determined. The results showed that the novel combination therapy stimulated anti-tumor immunity while retaining the ability to inhibit primary tumor growth of both murine colon (Colon26-HA) and mammary (4T1) carcinomas. The combination therapy resulted in enhanced tumor-specific T cell activation and controlled metastatic tumor growth. These results suggest that PDT may be an effective adjuvant for therapies that fail to stimulate the host anti-tumor immune response.

The effect of ephrin-A1 on resistance to Photofrin-mediated photodynamic therapy in esophageal squamous cell carcinoma cells.

Esophageal squamous cell carcinoma (ESCC), the most prevalent cell type of esophageal cancer, remains a dismal disease with poor prognosis. Photodynamic therapy (PDT) is a minimally invasive treatment option for early esophageal cancer. To explore possible factors involved in resistance to PDT in esophageal cancer cells, we selected PDT-resistant subcell lines by repeated treatment of CE48T/VGH (CE48T) ESCC cells with Photofrin-PDT and then analyzed the global gene modulations in the PDT-resistant cells by whole-genome microarray. More than 700 genes reached a fold change greater than 1.5 in each of the PDT-resistant cells compared to parental cells. Among these genes, both tumor necrosis factor (TNF) and EFNA1 genes were significantly upregulated in resistant cell lines. However, they were significantly downregulated in Photofrin-PDT-treated cells compared to untreated cells. The observations made in the microarray analysis were further confirmed by quantitative PCR. We observed that recombinant tumor necrosis factor alpha (TNF-α) activated the gene expression of EFNA1 at both the messenger RNA (mRNA) level and the protein level in CE48T cells. Functional analysis showed that when incubated with oligomeric and monomeric ephrin-A1 simultaneously, ESCC cells became significantly resistant to Photofrin-PDT. Functional analysis further suggested that transmembrane and soluble ephrin-A1 may cooperate to enhance resistance to Photofrin-PDT in ESCC cells.

Enhanced apoptotic effects by downregulating Mcl-1: evidence for the improvement of photodynamic therapy with Celecoxib.

Tumor cells exposed to sub-lethal photodynamic therapy (PDT) cause cellular rescue responses that lead to resistance to the therapy, including expression of angiogenic factors and survival molecules. However, the mechanisms contributing to the resistance are yet to be fully understood. Here, we show for the first time that Mcl-1, an anti-apoptotic protein, plays an important role in protecting cells from PDT-induced apoptosis. In contrast to the reduction in the anti-apoptotic proteins Bcl-2 and Bcl-xl, sub-lethal PDT induces an increase in Mcl-1 expression. Silencing Mcl-1 sensitizes tumor cells to PDT-induced apoptosis, and ectopic expression of Mcl-1 significantly delays Bax translocation to mitochondria and inhibits caspase-3 activity following PDT. Mcl-1 expression is associated closely with activated AKT signaling following PDT. AKT can regulate Mcl-1 expression through GSK-3ß and NF-κB at the protein and transcriptional levels, respectively. Inhibition of AKT by Wortmannin or siRNA significantly reduces the levels of Mcl-1 mRNA and protein and enhances PDT-induced apoptosis. Treatment with Celecoxib, a non-steroidal anti-inflammatory drug (NSAID), is shown to downregulate Mcl-1 expression, and enhances PDT-induced apoptosis both in vitro and in vivo. This down-regulation is closely related to the inhibition effect of Celecoxib on the AKT/GSK-3ß pathway, and was blocked upon addition of GSK-3ß inhibitor LiCl or the proteasome inhibitor MG132. These results suggest that Mcl-1 is a potential target for improving the antitumor efficiency of PDT. A loss in Mcl-1 by inhibiting AKT promotes PDT-induced apoptosis through the mitochondrial pathway. This also provides a novel rationale for utilizing Celecoxib to improve the efficacy of PDT.

Moraxella catarrhalis is susceptible to antimicrobial photodynamic therapy with Photofrin.

BACKGROUND AND OBJECTIVE: Moraxella catarrhalis is a significant cause of pediatric otitis media (OM), which is the most prevalent bacterial infection in children and primary reason for antibiotic administration in this population. Moreover, biofilm formation has been implicated as a primary mechanism of chronic or recurrent OM disease. As bacterial biofilms are inherently resistant to most antibiotics and these complex structures also present a significant challenge to the immune system, there is a clear need to identify novel antimicrobial approaches to treat OM infections. In this study, we evaluated the potential efficacy of antibacterial photodynamic therapy (aPDT) with porfimer sodium (Photofrin (PF)) against planktonic as well as biofilm-associated M. catarrhalis. MATERIALS AND METHODS: The bactericidal activity of aPDT with PF was assessed against multiple recent clinical isolates of M. catarrhalis grown planktonically as well as in biofilms. The bactericidal activity of PF-aPDT was quantified by enumeration of colony forming units post-treatment. The effect of aPDT on M. catarrhalis biofilms was further investigated with scanning electron microscopy (SEM) imaging. RESULTS: aPDT with PF significantly reduced M. catarrhalis viability. Although PF-aPDT caused higher killing in planktonic grown organisms (5-6 log kill), biofilm grown bacteria also demonstrated a statistically significant reduction in viable organisms (3-4 log decrease in recoverable bacteria) following treatment as compared to saline only controls (P < 0.01). SEM studies indicated the PF-aPDT treated bacteria exhibited prominent morphological changes with visibly distorted cell membranes. CONCLUSIONS: aPDT with PF elicits significant bactericidal activity against both planktonic and biofilm-associated M. catarrhalis, suggesting this technology warrants further analysis as a potential novel antimicrobial treatment for acute or recurrent OM.

The effects of Photofrin-mediated photodynamic therapy on the modulation of EGFR in esophageal squamous cell carcinoma cells.

Photodynamic therapy (PDT) has been demonstrated to be an effective minimally invasive treatment modality for early esophageal cancer. However, the molecular action in esophageal cancer during PDT is hardly known. EGFR has been known to downregulate in various cancer cells during PDT. In this study, we investigated the effects of Photofrin-mediated PDT on cell death and expression of EGFR in CE48T/VGH (CE48T) esophageal squamous cell carcinoma cells. We found that the photosensitizer Photofrin in the absence of light exposure can downregulate the expression of EGFR at both transcription and translation levels. Higher concentrations of Photofrin results in cytotoxicity whereas lower doses of Photofrin inhibit EGFR expression under dark control without inducing significant cell death. This Photofrin-associated inhibition of EGFR was repeated in lung cancer, cervical cancer, and glioblastoma cells. Another esophageal squamous cell carcinoma cell line CE81T/VGH (CE81T) was found to be resistant to Photofrin-induced inhibition of EGFR as well as to Photofrin-mediated dark toxicity compared with CE48T. The resistance to the cytotoxicity in CE81T cells became insignificant when the Photofrin-treated cells were further irradiated by red light (Photofrin-PDT). We suggest Photofrin modulates the expression of EGFR in cancer cells. However, efficient cell death still requires the combination of Photofrin and light irradiation in esophageal squamous cell carcinoma cells.

Combination with genistein enhances the efficacy of photodynamic therapy against human anaplastic thyroid cancer cells.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) has become one of the emerging options in management of cancer and other diseases. The major goal of PDT is to kill cancer cell without causing any adverse effect to the normal cells. PDT in combination of different therapeutic agents is being evaluated to improve the efficacy of treatment. Genistein, a soy ingredient, has widely been studied against different types of cancer. In the present study, combination of these two therapeutic methods has been studied to evaluate the enhanced effectiveness and find out the mechanism of action. MATERIALS AND METHODS: Combination of PDT and genistein has been studied against human thyroid cancer cells SNU 80. Cells were treated with genistein and different concentration of photofrin in PDT singly and conjointly. Viability of SNU 80 cells was analyzed using MTT assay. The cells were stained with Hoechst 33342 and propidium iodide (PI) for morphological observations. Changes in mitochondrial membrane potential and generation of reactive oxygen species (ROS) were also studied by confocal microscopy. Western blot analysis were also performed to find out the expressions of different pro- and anti-apoptotic proteins. RESULTS: From the result, the combination of genistein and photofrin mediated PDT enhanced the apoptotic effect against SNU 80 cells. Proliferation of the cells was inhibited and the mitochondrial membrane depolarisation was observed. ROS level were also increased in combination treatment. The expressions of Caspase 3, Caspase 9, cytochrome c, Caspase 8, Caspae 12 and other apoptosis related proteins were also modified. CONCLUSION: Thus PDT can induce apoptosis in thyroid cancer cells singly or in combination with genistein. But in combination treatment, the efficacy of inducing apoptosis in SNU 80 cells is much higher than that of individual treatment with genistein or PDT.

Photodynamic therapy as an alternative treatment for disinfection of bacteria in oral biofilms.

BACKGROUND AND OBJECTIVES: Biofilm-related diseases such as caries and periodontal disease are prevalent chronic oral infections which pose significant oral and general health risks. Biofilms are sessile communities attached to surfaces. Photodynamic therapy (PDT) has been demonstrated to have a significant anti-microbial effect and presents as an alternative to treating biofilm-related disease. The aim of this study was to determine the ability of porfimer sodium induced PDT to treat localized infections of Streptococcus mutans in biofilm communities. MATERIALS AND METHODS: Reproducible biofilms were formed by S. mutans strain ATCC 27351 growing in log phase at 37°C in Brain Heart Infusion medium, circulating through flow cells at 3 ml/minute for 36-48 hours. The photosensitizer used was porfimer sodium (Photofrin®) at 125 µg/ml with biofilm immersion times of 5 minutes and increasing energy density of post-immersion laser illumination at 630 nm (100 mW/cm(2) ). Resulting effects on bacterial viability in the biofilms were tracked by monitoring alamarBlue® conversion. Supplementary data characterizing the biofilms before and after exposure to PDT were acquired by Multiple Attenuated Internal Reflection Infrared Spectroscopy (MAIR-IR). RESULTS: The results of this study show that PDT using porfimer sodium and 630 nm laser light was effective in significantly reducing the viability of S. mutans biofilms. Maximum effectiveness was seen when biofilms were exposed to both photosensitizer and light versus controls. Porfimer sodium incubation times as short as 5 minutes in solutions as dilute as 25 µg/ml and illuminated with as little as 30 J/cm(2) resulted in significant decreases in viability of bacteria in biofilms. Optimum parameters appear to be 125 µg/ml porfimer sodium concentration and incubated for 5 minutes and 60 J/cm(2) of light energy density. CONCLUSIONS: This study has demonstrated that significant killing of the cariogenic organism S. mutans by the combination of a photosensitizer and the appropriate wavelength of laser light was possible even when the bacteria are embedded in an extracellular matrix.

Mechanism of mitochondrial membrane permeabilization during apoptosis under photofrin-mediated photodynamic therapy.

Photofrin-mediated photodynamic therapy (PF-PDT) can induce cell apoptosis via the mitochondria/caspase-3 pathway. Here, we further investigate the mechanism involved in the mitochondrial apoptotic process induced by PF-PDT. A high-level intracellular reactive oxygen species (ROS) generation in mitochondria, mitochondrial swelling, and dissipation of mitochondrial transmembrane potential were observed immediately after irradiation, indicating that mitochondria were the major ROS generation sites and also the first oxidative damage sites after PF-PDT treatment. For mitochondrial permeability detection, the decrease of calcein fluorescence emission intensity and release of cytochrome c were observed immediately after PF-PDT treatment, indicating the occurrence of mitochondrial inner membrane permeabilization (MIMP) and the mitochondrial outer membrane permeabilization (MOMP). However, cytochrome c release was not prevented by cyclosporine (CsA), a specific inhibitor of mitochondrial permeability transition (MPT). Taken together, these results demonstrated that PF-PDT caused simultaneous onset of MIMP and MOMP immediately after the treatment, and MOMP was independent of the MPT. Besides, inducible mitochondrial ROS generation played key roles in PF-PDT-induced cell apoptosis. This study will be benefit for understanding the mechanism involved in the initial mitochondrial oxidative damage by PF-PDT.

Evaluation of the Antitumor Immune Response Following Photofrin-Based PDT in Combination with the Epigenetic Agent 5-Aza-2'-Deoxycytidine.

Photofrin-based photodynamic therapy (PDT) is approved for clinical use by the US Food and Drug Administration and the European Medicines Agency and is among the most widely used photosensitizer for the treatment of cancer. It was broadly reported that both the innate and the adaptive arms of immune response can be activated by PDT and play a critical role in the anticancer outcome of this treatment. PDT leads to the induction of acute local inflammation that includes leukocyte infiltration as well as increased activation and production of pro-inflammatory factors and cytokines. These events can lead to the development of systemic and specific antitumor immune response. Combining Photofrin-PDT with the epigenetic agent 5-aza-2'-deoxycytidine results in potentiated antitumor effects in vivo. Understanding the molecular mechanisms underlying this phenomenon would be invaluable for clinical development of this therapeutic approach. This chapter describes a detailed protocol allowing evaluation of specific antitumor immune response induced by PDT.

Bax is essential for Drp1-mediated mitochondrial fission but not for mitochondrial outer membrane permeabilization caused by photodynamic therapy.

Bcl-2 family proteins are critical for the regulation of apoptosis, with the pro-apoptotic members Bax essential for the release of cytochrome c from mitochondria in many instances. However, we found that Bax was activated after mitochondrial depolarization and the completion of cytochrome c release induced by photodynamic therapy (PDT) with the photosensitizer Photofrin in human lung adenocarcinoma cells (ASTC-a-1). Besides, knockdown of Bax expression by gene silencing had no effect on mitochondrial depolarization and cytochrome c release, indicating that Bax makes no contribution to mitochondrial outer membrane permeabilization (MOMP) following PDT. Further study revealed that Bax knockdown only slowed down the speed of cell death induced by PDT, indicating that Bax is not essential for PDT-induced apoptosis. The fact that Bax knockdown totally inhibited the mitochondrial accumulation of dynamin-related protein (Drp1) and Drp1 knockdown attenuated cell apoptosis suggest that Bax can promote PDT-induced apoptosis through promoting Drp1 activation. Besides, Drp1 knockdown also failed to inhibit PDT-induced cell death finally, indicating that Bax-mediated Drp1's mitochondrial translocation is not essential for PDT-induced cell apoptosis. On the other hand, we found that protein kinase Cδ (PKCδ), Bim L and glycogen synthase kinase 3ß (GSK3ß) were activated upon PDT treatment and might contribute to the activation of Bax under the condition. Taken together, Bax activation is not essential for MOMP but essential for Drp1-mediated mitochondrial fission during the apoptosis caused by Photofrin-PDT.

Mono- and tri-cationic porphyrin-monoclonal antibody conjugates: photodynamic activity and mechanism of action.

Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.

Singlet oxygen is essential for neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.

Involvement of damage-associated molecular patterns in tumor response to photodynamic therapy: surface expression of calreticulin and high-mobility group box-1 release.

Damage-associated molecular patterns (DAMPs), danger signal molecules expressed after injury or infection, have become recognized as prerequisite for orchestrating effective anti-tumor host response. The expression of two prototypical DAMPs, calreticulin and high-mobility group box-1 (HMGB1) protein, was examined following Photofrin-photodynamic therapy (PDT) of Lewis lung carcinoma (LLC) cells in vitro and LLC tumors growing in syngeneic mice. Cell surface expression of calreticulin was found to be highly increased at 1 h after PDT treatment both in vitro and in vivo. Increased exposure of calreticulin was also detected on the surface of macrophages from PDT-treated LLC tumors. At the same time interval, a rise in serum HMGB1 was detected in host mice. Intracellular staining of macrophages co-incubated for 16 h with PDT-treated LLC cells revealed elevated levels of HMGB1 in these cells. The knowledge of the involvement of these DAMPs uncovers important mechanistic insights into the development of host response induced by PDT.

Molecular determinants of photodynamic therapy for lung cancers.

BACKGROUND AND OBJECTIVES: PDT induces apoptosis, inflammatory reactions, immune reactions, and damage to the microvasculature around the tumors. The mechanisms responsible for the anticancer effects of Photofrin-PDT and NPe6-PDT differ somewhat. To select a photosensitizer for lung cancer treatment and to improve the efficacy of PDT, the mechanisms of action for PDT using Photofrin or NPe6 must be elucidated and the phenomena validated by analyzing molecular determinants from clinical samples. STUDY DESIGN/MATERIALS AND METHODS: We examined the role of immunological reactions in the anti-tumor effects of PDT using cytokine-overexpressing cells and investigated whether the anti-apoptotic protein Bcl-2 may be a molecular target. Moreover, we investigated the association between ATP-binding cassette transporter proteins such as breast cancer-resistant protein (BCRP), which can pump out some types of photosensitizer, and the efficacy of PDT using clinical samples from 81 early lung cancer lesions treated with PDT between 1998 and 2006 at the Tokyo Medical University Hospital. RESULTS: Photofrin-PDT damaged Bcl-2 and rapidly induced apoptosis, but NPe6-PDT did not damage Bc-2 nor did it induce morphologically typical apoptosis. However, NPe6-PDT exerted a strong anti-tumor effect, regardless of the overexpression of Bcl-2. By analyzing the BCRP-overexpressing cells, Photofrin, but not NPe6, was found to be a substrate of BCRP. All 81 lung cancer lesions were BCRP-positive; as Photofrin was found to be a substrate of BCRP, the expression of BCRP significantly affected the efficacy of Photofrin-PDT. However, NPe6-PDT exerted a strong antitumor effect regardless of BCRP expression, and the complete response rate after NPe6-PDT was much higher than that after Photofrin-PDT. CONCLUSIONS: Our translational research suggests that NPe6-PDT may be superior to Photofrin-PDT for the treatment of lung caner, and individualized approaches to PDT based on the expression status of Bcl-2 and/or BCRP may improve the efficacy of PDT in patients with lung cancers.

Pro-apoptotic and anti-inflammatory properties of the green tea constituent epigallocatechin gallate increase photodynamic therapy responsiveness.

BACKGROUND: A polyphenol constituent of green tea, epigallocatechin gallate (EGCG), has anti-carcinogenic properties. A growing number of studies document EGCG-mediated induction of apoptotic pathways and inhibition of pro-survival factors when combined with chemotherapy or radiation. We evaluated the efficacy of EGCG in modulating photofrin (PH)-mediated photodynamic therapy (PDT) responses. METHODS: Mouse mammary carcinoma (BA) cells and transplanted BA tumors growing in C3H mice were treated with PH-mediated PDT. Select groups of treated cells and mice also received EGCG and then cytotoxicity, tumor response, and expression of survival molecules were evaluated in all experimental groups. RESULTS: EGCG increased apoptosis and cytotoxicity in BA cells exposed to PH-mediated PDT. The initial pro-survival phase of the unfolded protein response (UPR), characterized by increased expression of the 78 kDa glucose-regulated protein (GRP-78), was induced by PDT. The second pro-apoptotic phase of the UPR, characterized by phospho-c-Jun N-terminal kinase (p-JNK) expression, activation of caspases-3 and 7, poly ADP ribose polymerase (PARP) cleavage, and expression of C/EBP homologous protein was observed when PDT was combined with EGCG. EGCG also decreased the expression of the pro-survival proteins GRP-78 and survivin, and attenuated PDT-induced prostaglandin E2 (PGE2 ) expression in PDT-treated cells. Comparable responses also were observed when BA tumors were treated with PDT and EGCG. In addition, PDT-induced expression of metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) was down-regulated in treated tumor tissue by EGCG. CONCLUSIONS: The polyphenol EGCG improves PDT efficacy by increasing tumor apoptosis and decreasing expression of pro-survival and angiogenic molecules within the tumor microenvironment.

IL-6 potentiates tumor resistance to photodynamic therapy (PDT).

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) is an anticancer modality approved for the treatment of early disease and palliation of late stage disease. PDT of tumors results in the generation of an acute inflammatory response. The extent and duration of the inflammatory response is dependent upon the PDT regimen employed and is characterized by rapid induction of proinflammatory cytokines, such as IL-6, and activation and mobilization of innate immune cells. The importance of innate immune cells in long-term PDT control of tumor growth has been well defined. In contrast the role of IL-6 in long-term tumor control by PDT is unclear. Previous studies have shown that IL-6 can diminish or have no effect on PDT antitumor efficacy. STUDY DESIGN/MATERIALS AND METHODS: In the current study we used mice deficient for IL-6, Il6(-/-) , to examine the role of IL-6 in activation of antitumor immunity and PDT efficacy by PDT regimens known to enhance antitumor immunity. RESULTS: Our studies have shown that elimination of IL-6 had no effect on innate cell mobilization into the treated tumor bed or tumor draining lymph node (TDLN) and did not affect primary antitumor T-cell activation by PDT. However, IL-6 does appear to negatively regulate the generation of antitumor immune memory and PDT efficacy against murine colon and mammary carcinoma models. The inhibition of PDT efficacy by IL-6 appears also to be related to regulation of Bax protein expression. Increased apoptosis was observed following treatment of tumors in Il6(-/-) mice 24 hours following PDT. CONCLUSIONS: The development of PDT regimens that enhance antitumor immunity has led to proposals for the use of PDT as an adjuvant treatment. However, our results show that the potential for PDT induced expression of IL-6 to enhance tumor survival following PDT must be considered.

Novel applications of diagnostic X-rays in activating a clinical photodynamic drug: Photofrin II through X-ray induced visible luminescence from "rare-earth" formulated particles.

INTRODUCTION: In this communication we report on a novel non-invasive methodology in utilizing "soft" energy diagnostic X-rays to indirectly activate a photo-agent utilized in photodynamic therapy (PDT): Photofrin II (Photo II) through X-ray induced luminescence from Gadolinium Oxysulfide (20 micron dimension) particles doped with Terbium: Gd_{2}O_{2}S:Tb. Photodynamic agents such as Photo II utilized in PDT possess a remarkable property to become preferentially retained within the tumor's micro-environment. Upon the photo-agent's activation through (visible light) photon absorption, the agents exert their cellular cytotoxicity through type I and type II pathways through extensive generation of reactive oxygen species (ROS); namely, singlet oxygen ^{1}O_{2}, superoxide anion O_{2}^{-}, and hydrogen peroxide H_{2}O_{2}, within the intra-tumoral environment. Unfortunately, due to shallow visible light penetration depth (∼ 2 mm to 5 mm) in tissues, the current PDT strategy has largely been restricted to the treatment of surface tumors, such as the melanomas. Additional invasive strategies through optical fibers are currently utilized in getting the visible light into the intended deep seated targets within the body for PDT. METHODS: X-ray induced visible luminescence from Gd_{2}O_{2}S:Tb particles were spectroscopically characterized, and the potential in-vitro cellular cytotoxicity of Gd_{2}O_{2}S:Tb particles on human glioblastoma cells (due to 48 Hrs Gd_{2}O_{2}S:Tb particle exposure) was screened through the MTS cellular metabolic assay. In-vitro human glioblastoma cellular exposures in presence of Photo II with Gd_{2}O_{2}S:Tb particles were performed in the dark in sterile 96 well tissue culture plates, and the corresponding changes in the metabolic activities of the glioblastoma due to 15 minutes of (diagnostic energy) X-ray exposure was determined 48 Hrs after treatment through the MTS assay. RESULTS: Severe suppression (> 90% relative to controls) in the cellular metabolic activity of human glioblastoma was measured due to the treatment of clinically relevant concentrations of 20 µg/ml Photo II, with Gd_{2}O_{2}S:Tb particles, and (120 kVp) diagnostic X-rays. Taken together, the in-vitro findings herein provide the basis for future studies in determining the safety and efficacy of this non-invasive X-ray induced luminescence strategy in activating photo-agent in deep seated tumors.

Inhibitive effects of photofrin on cellular autophagy.

Depending on the circumstances, autophagy can be either a protective or damaging cellular process. The role of autophagy in photodynamic therapy (PDT), a photo-chemotherapy that utilizes light to activate a photosensitizer drug to achieve localized cellular damage, has been explored in recent years. It has been reported that autophagy in PDT is significantly influenced by the treatment protocol. In this work, the role of Photofrin, a well-established clinical photosensitizer, in regulating cellular autophagy was investigated. The effects of Photofrin on cellular autophagy induced by conventional starvation or rapamycin techniques were studied. By fluorescence imaging, Western blotting and cell viability assays, it was found that Photofrin can effectively inhibit cellular autophagy induced by starvation or rapamycin. This autophagy blocking is independent of the photosensitizing property of the drug. With Baf-A1, a well-established agent that inhibits autophagosome from fusing with lysosome, we also found that, the observed phenomenon is not due to accelerated degradation of existing autophagosomes, thus proving that the drug Photofrin alone, without light excitation, can truly block autophagy.