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.

Verteporfin- and sodium porfimer-mediated photodynamic therapy enhances pancreatic cancer cell death without activating stromal cells in the microenvironment.

The goal of our study was to determine the susceptibility of different pancreatic cell lines to clinically applicable photodynamic therapy (PDT). The efficacy of PDT of two different commercially available photosensitizers, verteporfin and sodium porfimer, was compared using a panel of four different pancreatic cancer cell lines, PANC-1, BxPC-3, CAPAN-2, and MIA PaCa-2, and an immortalized non-neoplastic pancreatic ductal epithelium cell line, HPNE. The minimum effective concentrations and dose-dependent curves of verteporfin and sodium porfimer on PANC-1 were determined. Since pancreatic cancer is known to have significant stromal components, the effect of PDT on stromal cells was also assessed. To mimic tumor-stroma interaction, a co-culture of primary human fibroblasts or human pancreatic stellate cell (HPSCs) line with PANC-1 was used to test verteporfin-PDT-mediated cell death of PANC-1. Two cytokines (TNF-α and IL-1ß) were used for stimulation of primary fibroblasts (derived from human esophageal biopsies) or HPSCs. The increased expression of smooth muscle actin (α-SMA) confirmed the activation of fibroblasts or HPSC upon treatment with TNF-α and IL-1ß. Cell death assays showed that both sodium porfimer- and verteporfin-mediated PDT-induced cell death in a dose-dependent manner. However, verteporfin-PDT treatment had a greater efficiency with 60 × lower concentration than sodium porfimer-PDT in the PANC-1 incubated with stimulated fibroblasts or HPSC. Moreover, activation of stromal cells did not affect the treatment of the pancreatic cancer cell lines, suggesting that the effects of PDT are independent of the inflammatory microenvironment found in this two-dimensional culture model of cancers.

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.

Biological effects in photodynamic treatment combined with electropermeabilization in wild and drug resistant breast cancer cells.

Electrochemotherapy became one of the therapeutic protocols successfully used in oncology. However, biological effects occurring in cells, especially those which are drug resistant, have not been studied thoroughly. This study presents response of wild and drug resistant breast cancer cells to classical photodynamic therapy with Photofrin or experimental photodynamic therapy with cyanine IR-775, combined with electroporation. Photodynamic reaction or electroporation alone had no cytotoxic effect, but their combination significantly disturbed cellular functions. Applying electroporation allowed the drugs to increase its accumulation, especially for a poorly permeant cyanine in drug resistant cells. FACS analysis showed that even at relatively mild electric field, ca. 90% of cells were permeabilized. High intracellular concentration of drugs triggered the cellular defense system through increased expression of glutathione S-transferase and multidrug resistance proteins (MDR1 and MRP7), particularly in drug resistant cells. Finally, expressively decreased cell metabolism and proliferation, as well as formation of apoptotic bodies and fragmentation of cells were observed after the combined treatment. The results show that electroporation can be used for effective delivery of photosensitizers, even to drug resistant breast cancer cells, which was not tested before. This shows that electro-photodynamic treatment could be a promising approach to overcome a problem of drug resistance in cancer cells.

Enhanced production of reactive oxygen species in HeLa cells under concurrent low­dose carboplatin and Photofrin® photodynamic therapy.

Concurrent low­dose carboplatin/Photofrin® photodynamic therapy (ccPDT) has been shown to promote relapse­free complete tumor regression in cervical or endometrial cancer patients as a fertility­preservation therapy. This study aimed to investigate the molecular mechanism of the enhanced therapeutic efficacy of ccPDT by determining intracellular reactive oxygen species (ROS) and necrotic or apoptotic cell damage in HeLa cells loaded with fluorescent oxidant agents and Photofrin or/and carboplatin under light irradiation. The cytotoxic effects of ccPDT were compared when monitored with a light dose under carboplatin or Photofrin alone. Photofrin­PDT alone did not enhance either hydroxyl radicals (OH•) or superoxide anions (O2•-), but a slight enhancement of hydrogen peroxide (H2O2) production was observed. A larger enhancement of ROS production was obtained in a dose­dependent manner following ccPDT, especially OH• and H2O2, in conjunction with both necrotic and apoptotic cell death, compared with necrotic­prone PDT alone. The carboplatin­mediated Fenton reaction: 2[PtII]2 + H2O2 → [Pt2.25]4 + OH¯+ OH• was proposed to explain the dose­dependent enhancement of OH•. In conclusion, the therapeutic enhancement of ccPDT in vitro was attributable to the carboplatin­mediated synergetic production of OH▪ and apoptotic cellular damage, compared with Photofrin­PDT alone.

Photodynamic therapy does not induce cyclobutane pyrimidine dimers in the presence of melanin.

Photodynamic therapy (PDT) is an office-based treatment for precancerous and early cancerous skin changes. PDT induces cell death through the production of reactive oxygen species (ROS). Cyclobutane pyrimidine dimers (CPDs) are the most important DNA changes responsible for ultraviolet (UV) carcinogenesis. Recently ROS induced by UVA were shown to generate CPDs via activating melanin. This raised the possibility that PDT induced ROS may also induce CPDs and mutagenesis in melanin containing cells. Previously the effect of PDT on CPDs in melanin containing cells has not been assessed. Our current work aimed to compare the generation of CPDs in melanin containing cells subjected to UVA treatment and porfimer sodium red light PDT. We used ELISA to detect CPDs. After UVA we found a dose dependent increase in CPDs in melanoma cells (B16-F10, MNT-1) with CPD levels peaking hours after discontinuation of UVA treatment. This indicated the generation of UVA induced dark-CPDs in the model. Nevertheless, PDT in biologically relevant doses was unable to induce CPDs. Our work provides evidence for the lack of CPD generation by PDT in melanin containing cells.

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.

Sulforaphene Enhances The Efficacy of Photodynamic Therapy In Anaplastic Thyroid Cancer Through Ras/RAF/MEK/ERK Pathway Suppression.

Sulforaphene (SFE), a natural isothiocyanate from cruciferous vegetables has shown a potential anticancer effect against cervical and lung cancer. Palliative treatments like photodynamic therapy (PDT) are being implemented for a long time however, the results are still not promising in case of aggressive cancers like anaplastic thyroid cancer. The objective of this work is to establish an alternative method with the combination of photofrin-PDT and sulforaphene, a natural isothiocyanate from cruciferous vegetables, against human anaplastic thyroid cancer to enhance the efficacy of PDT. In this study, cell viability of FRO cells due to combination treatment was analyzed by MTT assay, Cell cycle arrest, MMP depolarization and ROS generation, analyzed by flow cytometry. Western blot analysis of various proliferative proteins was performed to assess the activity of combination treatment against FRO cells. From the results, sulforaphene alone showed no cytotoxicity against normal cells, however, combination of sulforaphene and photofrin mediated PDT showed a noticeable decrease in cell proliferation against FRO cells. Combination treatment synergistically caused cell cycle arrest via ROS generation and MMP depolarization. The expressions of Ras, MEK, ERK, B-Raf proteins significantly modulated due to combination treatment. PDT and SFE can induce apoptosis in anaplastic thyroid cancer cells individually but while treated in combination, it enhanced the apoptotic and anti-proliferative effect, much higher than the individual doses. In summary, our work designates sulforaphene as a unique natural enhancer of efficacy with PDT against anaplastic thyroid cancer.

Enhanced phototoxicity of photodynamic treatment by Cx26-composed GJIC via ROS-, calcium- and lipid peroxide-mediated pathways.

In spite of the promising initial treatment responses presented by photodynamic therapy (PDT), 5-year recurrence rates remain high level. Therefore, improvement in the efficacy of PDT is needed. There are reports showing that connexin(Cx) 26-composed gap junctional intercellular communication (GJIC) enhances the intercellular propagation of "death signal", thereby increasing chemotherapeutic cytotoxicity. However, it is unclear whether Cx26-formed GJIC has an effect on PDT phototoxicity. The results in the present study showed that Cx26-composed GJ formation at high density enhances the phototoxicity of Photofrin-PDT. When the Cx26 is not expressed or Cx26 channels are blocked, the phototoxicity in high-density cultures substantially reduces, indicating that the enhanced PDT phototoxicity at high density is mediated by Cx26-composed GJIC. The GJIC-mediated increase in PDT phototoxicity was associated with ROS, calcium and lipid peroxide-mediated stress signaling pathways. The work presents the ability of Cx26-composed GJIC to enhance the sensitivity of malignant cells to PDT, and indicates that maintenance or increase of Cx26-formed GJIC may be a profitable strategy towards the enhancement of PDT therapeutic efficiency. Picture: The survival response of Photofrin-PDT in Dox-treated (Cx26 expressing, GJ-formed) and Dox-untreated cells (Cx26 non-expressing, GJ-unformed) at high-cell density condition.

Potassium Iodide Potentiates Broad-Spectrum Antimicrobial Photodynamic Inactivation Using Photofrin.

It is known that noncationic porphyrins such as Photofrin (PF) are effective in mediating antimicrobial photodynamic inactivation (aPDI) of Gram-positive bacteria or fungi. However, the aPDI activity of PF against Gram-negative bacteria is accepted to be extremely low. Here we report that the nontoxic inorganic salt potassium iodide (KI) at a concentration of 100 mM when added to microbial cells (108/mL) + PF (10 µM hematoporphyrin equivalent) + 415 nm light (10 J/cm2) can eradicate (>6 log killing) five different Gram-negative species (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter baumannii), whereas no killing was obtained without KI. The mechanism of action appears to be the generation of microbicidal molecular iodine (I2/I3-) as shown by comparable bacterial killing when cells were added to the mixture after completion of illumination and light-dependent generation of iodine as detected by the formation of the starch complex. Gram-positive methicillin-resistant Staphylococcus aureus is much more sensitive to aPDI (200-500 nM PF), and in this case potentiation by KI may be mediated mainly by short-lived iodine reactive species. The fungal yeast Candida albicans displayed intermediate sensitivity to PF-aPDI, and killing was also potentiated by KI. The reaction mechanism occurs via singlet oxygen (1O2). KI quenched 1O2 luminescence (1270 nm) at a rate constant of 9.2 × 105 M-1 s-1. Oxygen consumption was increased when PF was illuminated in the presence of KI. Hydrogen peroxide but not superoxide was generated from illuminated PF in the presence of KI. Sodium azide completely inhibited the killing of E. coli with PF/blue light + KI.

Subcellular Targeting as a Determinant of the Efficacy of Photodynamic Therapy.

In prior studies, we have identified the ability of low-level lysosomal photodamage to potentiate the phototoxic effect of subsequent photodamage to mitochondria. The mechanism involves calpain-mediated cleavage of the autophagy-associated protein ATG5 to form a proapoptotic fragment (tATG5). In this report, we explore the permissible time lag between the two targeting procedures along with the effect of simultaneously targeting both lysosomes and mitochondria. This was found to be as effective as the sequential protocol with no gap between the irradiation steps. Inhibition of calpain reversed the enhanced efficacy of the "simultaneous" protocol. It appears that even a minor level of lysosomal photodamage can have a significant effect on the efficacy of subsequent mitochondrial photodamage. We propose that these results may explain the efficacy of Photofrin, a photosensitizing product that also targets both lysosomes and mitochondria for photodamage.

Low dose of GRP78-targeting subtilase cytotoxin improves the efficacy of photodynamic therapy in vivo.

Photodynamic therapy (PDT) exerts direct cytotoxic effects on tumor cells, destroys tumor blood and lymphatic vessels and induces local inflammation. Although PDT triggers the release of immunogenic antigens from tumor cells, the degree of immune stimulation is regimen-dependent. The highest immunogenicity is achieved at sub-lethal doses, which at the same time trigger cytoprotective responses, that include increased expression of glucose-regulated protein 78 (GRP78). To mitigate the cytoprotective effects of GRP78 and preserve the immunoregulatory activity of PDT, we investigated the in vivo efficacy of PDT in combination with EGF-SubA cytotoxin that was shown to potentiate in vitro PDT cytotoxicity by inactivating GRP78. Treatment of immunocompetent BALB/c mice with EGF-SubA improved the efficacy of PDT but only when mice were treated with a dose of EGF-SubA that exerted less pronounced effects on the number of T and B lymphocytes as well as dendritic cells in mouse spleens. The observed antitumor effects were critically dependent on CD8+ T cells and were completely abrogated in immunodeficient SCID mice. All these results suggest that GRP78 targeting improves in vivo PDT efficacy provided intact T-cell immune system.

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.

Singlet oxygen treatment of tumor cells triggers extracellular singlet oxygen generation, catalase inactivation and reactivation of intercellular apoptosis-inducing signaling.

Intracellular singlet oxygen generation in photofrin-loaded cells caused cell death without discrimination between nonmalignant and malignant cells. In contrast, extracellular singlet oxygen generation caused apoptosis induction selectively in tumor cells through singlet oxygen-mediated inactivation of tumor cell protective catalase and subsequent reactivation of intercellular ROS-mediated apoptosis signaling through the HOCl and the NO/peroxynitrite signaling pathway. Singlet oxygen generation by extracellular photofrin alone was, however, not sufficient for optimal direct inactivation of catalase, but needed to trigger the generation of cell-derived extracellular singlet oxygen through the interaction between H2O2 and peroxynitrite. Thereby, formation of peroxynitrous acid, generation of hydroxyl radicals and formation of perhydroxyl radicals (HO2(.)) through hydroxyl radical/H2O2 interaction seemed to be required as intermediate steps. This amplificatory mechanism led to the formation of singlet oxygen at a sufficiently high concentration for optimal inactivation of membrane-associated catalase. At low initial concentrations of singlet oxygen, an additional amplification step needed to be activated. It depended on singlet oxygen-dependent activation of the FAS receptor and caspase-8, followed by caspase-8-mediated enhancement of NOX activity. The biochemical mechanisms described here might be considered as promising principle for the development of novel approaches in tumor therapy that specifically direct membrane-associated catalase of tumor cells and thus utilize tumor cell-specific apoptosis-inducing ROS signaling.

Oxidative modification induced by photodynamic therapy with Photofrin®II and 2-methoxyestradiol in human ovarian clear carcinoma (OvBH-1) and human breast adenocarcinoma (MCF-7) cells.

Ovarian cancer is among the most lethal cancers in women. The successful anticancer treatment depends on the effectiveness of cytotoxic effect of applied therapeutic procedures either alone or in combination with other treatments. Photodynamic therapy (PDT) is a relatively new method of anticancer therapy. Its dominant mechanism of action is the over-production of reactive oxygen species induced by oxidative stress in malignant cells, which attack lipid membranes, proteins and nucleic acids. One of the important mechanisms is induction of unfolded protein response, ubiquitin-proteasome pathway of protein degradation. The aim of this study was to evaluate the cytotoxic effect of various protective enzymes in ovarian carcinoma clear cell line in comparison to the model breast cell line after photodynamic reaction and photodynamic reaction with 2-methoxyestradiol (2-Me). Human malignant ovarian cell line (OvBH-1) was used and human breast adenocarcinoma cells (MCF-7) were used as a control. Photodynamic reaction (PDR) with Photofrin(®)II and Ph(®)II with 2-Me was performed. The expression of protective proteins by immunocytochemistry (HSP70 and iNOS) and western blot (Hsp27 and Hsp70) methods was evaluated directly, 3 and 6 h after PDR. The changes in cells' cytoskeleton were evaluated using immunofluorescence by confocal microscopy. The expression of iNOS was observed for both experiments with differential intensity and quantity. A higher expression of Hsp70 in MCF-7 cells was observed than in OvBh-1 cells. The reorganization of cytoskeleton and nucleus was observed after 3 and 6 h after exposition to light.

Effects of electrophotodynamic therapy in vitro on human melanoma cells--melanotic (MeWo) and amelanotic (C32).

Photodynamic therapy has been considered ineffective for melanomas because of the competition between the absorbance of melanin from the melanoma and the absorbance of photosensitizers at the photosensitizer excitation light wavelength. Melanomas show considerable heterogeneity and resistance to phototherapy. The effectiveness of photodynamic therapy could be intensified by electroporation for enhanced transport of a photosensitizer by transient pores in the membrane. In this study, photodynamic therapy combined with electroporation was tested in vitro on the human melanoma cell lines melanotic melanoma (MeWo) and amelanotic melanoma (C32). Control experiments were conducted on human keratinocytes (HaCaT). Photofrin was used as a photosensitizer. Photosensitizer distribution, cloning efficacy test, comet assay, and assessment of apoptotic proteins were performed. Melanin levels were determined before and after photodynamic therapy. The experiments indicated that electroporation effectively supports the photodynamic method. It was found that photodynamic therapy with electroporation efficiently induces apoptosis in melanotic and amelanotic melanoma cells.

GJIC Enhances the phototoxicity of photofrin-mediated photodynamic treatment by the mechanisms related with ROS and Calcium pathways.

Despite initially positive responses, recurrences after Photodynamic treatment (PDT) can occur and there is need for improvement in the effectiveness of PDT. Our study uniquely showed that there was a significantly gap junctional intercellular communication (GJIC)-dependent PDT cytotoxicity. The presence of GJIC composed of Connexin 32 increased the PDT phototoxicity in transfected HeLa cells and in the xenograft tumors, and the enhanced phototoxicity of Photofrin-mediated PDT by GJIC was related with ROS and calcium pathways. Our study indicates the possibility that up-regulation or maintenance of gap junction functionality may be used to increase the efficacy of PDT. The phototoxicity effect of Photofrin was substantially greater in Dox-treated cells, which expressed the Cx32 and formed the GJ, than Dox-untreated.

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.

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.

GRP78-targeting subtilase cytotoxin sensitizes cancer cells to photodynamic therapy.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.