Photodynamic Therapy Induces Cell Death Research Articles

The Potential of Photodynamic Therapy Using Solid Lipid Nanoparticles with Aluminum Phthalocyanine Chloride as a Nanocarrier for Modulating Immunogenic Cell Death in Murine Melanoma In Vitro.

Photodynamic therapy (PDT) uses a photosensitizer to generate reactive oxygen species (ROS) that kill target cells. In cancer treatments, PDT can potentially induce immunogenic cell death (ICD), which is characterized by a well-controlled exposure of damage-associated molecular patterns (DAMPs) that activate dendritic cells (DCs) and consequently modulate the immune response in the tumor microenvironment. However, PDT still has limitations, such as the activity of photosensitizers in aqueous media and poor bioavailability. Therefore, a new photosensitizer system, SLN-AlPc, has been developed to improve the therapeutic efficacy of PDT. In vitro experiments showed that the light-excited nanocarrier increased ROS production in murine melanoma B16-F10 cells and modulated the profile of DCs. PDT induced cell death accompanied by the exposure of DAMPs and the formation of autophagosomes. In addition, the DCs exposed to PDT-treated B16-F10 cells exhibited morphological changes, increased expression of MHCII, CD86, CD80, and production of IL-12 and IFN-γ, suggesting immune activation towards an antitumor profile. These results indicate that the SLNs-AlPc protocol has the potential to improve PDT efficacy by inducing ICD and activating DCs.

Nanobody targeted photodynamic therapy induces cell death of HER2-positive tumor cells

SignificanceHuman epidermal growth factor receptor 2 (HER2) protein promotes the growth of cancer cells. Photodynamic therapy (PDT) using nanobodies (NBs) targeting HER2 is a promising treatment strategy for patients with HER2-positive cancers. ApproachIn this study, we evaluated the affinity of pheophorbide A (Pa)-conjugated HER2-targeted NBs (Pa-NB) conjugates for HER2-positive cells and the cytotoxicity induced by PDT using Pa-NB conjugates in vitro. MCF-7 cells with low HER2 expression and SKBR-3 with high HER2 expression were used. ResultsPa-NB conjugates had e high affinity for SKBR-3 cells and selectively induced cell death in them after laser irradiation. Pa-NB conjugates selectively accumulated in vitro. Upon laser irradiation, the Pa-NB conjugates selectively induced significant cytotoxicity in HER2-positive cells. ConclusionsPDT using Pa-NB conjugates, has been suggested as an attractive new therapeutic modality for HER2-positive cancers. Further studies are needed to assess the selectivity and antitumor effects of the Pa-NB conjugates in vivo.

Glucocorticoid receptor mediated sensitization of colon cancer to photodynamic therapy induced cell death

Photodynamic therapy (PDT) is a clinically approved, non-invasive alternate cancer therapy. A synthetic glucocorticoid (GC), dexamethasone (Dex) has previously been demonstrated to sensitize cancer cells to chemotherapy. However, to the best of our knowledge, the sensitization effect of GCs on PDT has not yet been investigated. We hypothesized that glucocorticoid receptor (GR) targeting can selectively make cancer cells more sensitive to PDT treatment, as PDT induces hypoxia wherein GR-activity gets enhanced. In addition, Dex was reported to act against the PDT-induced cell survival pathways like HIF-1α, NRF2, NF-κB, STAT3 etc. Thus, both the treatments can complement each other and may result in increasing the effectiveness of combination therapy. Hence, in this study, we developed liposomal formulations of our previously reported PDT agent P-Nap, either alone (D1P-Nap) or in combination with Dex (D1XP-Nap) to elucidate the sensitization effect. Interestingly, our RT-PCR results in hypoxic conditions showed down-regulation of HIF-1α and over expression of GR-activated genes for glucose-6-phosphatase (G6Pase) and PEPCK enzymes, indicating prominent GR-transactivation. We also observed higher phototoxicity in CT26.WT cells treated with D1XP-Nap PDT under hypoxic conditions as compared to normoxic conditions. These effects were reversed when cells were pre-treated with RU486, a competitive inhibitor of GCs. Moreover, our in vivo findings of subcutaneous tumor model of Balb/C mice for colon cancer revealed a significant decrease in tumor volume as well as considerable enhancement in the survivability of PDT treated tumor-bearing mice when Dex was present in the formulation. A high Bax/Bcl-xL ratio, high p53 expression, enhanced E-cadherin expression and down-regulation of pro-tumorigenic transcription factors NF-κB and c-Myc were found in tumor lysates from mice treated with D1XP-Nap under PDT, indicating GR-mediated sensitization of the tumor to PDT-induced cell death and enhancement of life-span for tumor bearing mice.

Graphene quantum dots-loaded macrophages as a biomimetic delivery system for bioimaging and photodynamic therapy

Macrophages have been recommended for site-specific tumoral delivery of genes, drugs, or nanoparticles. Graphene quantum dots (GQDs) are a well-known fluorescence-based diagnosis and photodynamic therapy (PDT) agent. Herein, we determined the therapeutic efficacy of hybrid GQD-loaded macrophages in a 3D culture model and a xenograft mouse model of human lung cancer cells. We examined the PDT efficacy of hybrid GQD-loaded macrophages in both models using LED light. Cell viability assay showed that the LED light-based PDT induced cell death via GQDs. Reactive oxygen species and Ca2+ detection assays indicated that GQD-loaded macrophages induced apoptosis in the in vitro 3D culture model. In vivo fluorescence bioimaging and ex vivo fluorescence images of major organs (the kidney, liver, lung, spleen, and tumor) revealed a tumor-homing effect of GQD-loaded macrophages in the xenograft mouse model. Our results demonstrated that hybrid GQD-loaded macrophages are a promising biomimetic delivery system for bioimaging and cancer therapy.

In Vitro Comparative Study of Near-Infrared Photoimmunotherapy and Photodynamic Therapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a new phototherapy that utilizes a monoclonal antibody (mAb) against cancer antigens and a phthalocyanine dye, IRDye700DX (IR700) conjugate (mAb-IR700). Photodynamic therapy (PDT) is a combination therapy that utilizes photoreactive agents and light irradiation as well as NIR-PIT. In the present study, we compared these therapies in vitro. The characterization of cellular binding/uptake specificity and cytotoxicity were examined using two mAb-IR700 forms and a conventional PDT agent, talaporfin sodium, in three cell lines. As designed, mAb-IR700 had high molecular selectivity and visualized target molecule-positive cells at the lowest concentration examined. NIR-PIT induced necrosis and damage-associated molecular patterns (DAMPs), a surrogate maker of immunogenic cell death. In contrast, talaporfin sodium was taken up by cells regardless of cell type, and its uptake was enhanced in a concentration-dependent manner. PDT induced cell death, with the pattern of cell death shifting from apoptosis to necrosis depending on the concentration of the photosensitizer. Induction of DAMPs was observed at the highest concentration, but their sensitivity differed among cell lines. Overall, our data suggest that molecule-specific NIR-PIT may have potential advantages compared with PDT in terms of the efficiency of tumor visualization and induction of DAMPs.

Photodynamic Therapy Induced Cell Death Mechanisms in Breast Cancer.

Breast cancer is the second most common cancer globally and the pioneering cause of mortality among women. It usually begins from the ducts or lobules, referred to as ductal carcinoma in situ, or lobular carcinoma in situ. Age, mutations in Breast Cancer Gene 1 or 2 (BRCA1 or BRCA2) genes, and dense breast tissue are the highest risk factors. Current treatments are associated with various side effects, relapse, and a low quality of life. Although conventional treatments, such as surgery and chemotherapy, have been used for decades, their adverse side effects on normal cells and tissues pose a major weakness, which calls for a non-invasive treatment option. Photodynamic therapy (PDT) has proven to be a promising form of cancer therapy. It is less invasive, target-specific, and with reduced cytotoxicity to normal cells and tissues. It involves the use of a photosensitizer (PS) and light at a specific wavelength to produce reactive oxygen species. One of the reasons for the target specificity is associated with the dense vascularization of cancer tissues, which tends to increase the surface area for the PS uptake. Photosensitizers are light-sensitive molecules, which result in cancer cell destruction followed by light irradiation. Depending on the localization of the PS within the cancer cell, its destruction may be via apoptosis, necrosis, or autophagy. This review focuses on the breast cancer etiopathology and PDT-induced cell death mechanisms in breast cancer cells.

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.

Thermal Ultra Short Photodynamic Therapy: Heating Fibroblasts During Sub-30-Minute Incubation of 5-Aminolevulinic Acid Increases Photodynamic Therapy-Induced Cell Death.

Actinic keratoses (AKs) prevalence was estimated at 39.5 million Americans in 2004, and the cost to treat AKs that year was approximately 1 billion dollars. Photodynamic therapy (PDT) is an FDA-approved therapy for the treatment of AK. Recent studies have focused on reducing PDT treatment time while maintaining efficacy. To investigate the use of thermal modulation to improve the efficacy of ultra short aminolevulinic acid (ALA) incubation PDT. Human dermal fibroblasts (HDFs) were incubated for 10, 15, or 20 minutes with 0.5-mM ALA at various temperatures (21, 24, 27, 30, 33, 36, 39, and 42°C). After ALA incubation, samples were treated for 1,000 seconds with blue light (417 ± 5 nm) resulting in a fluence of 10 J/cm. Samples were collected and stained for apoptosis/necrosis with annexin-V and 7-aminoactinomycin D (7-AAD), then analyzed by flow cytometry. Human dermal fibroblast treated with 10-minute ALA-PDT had no statistically significant changes in apoptosis at all temperatures. Human dermal fibroblast treated with 15- or 20-minute ALA-PDT had statistically significant increases in apoptosis at 39 and 42°C (p < .05). These results suggest the use of thermal modulation may improve ultra short ALA incubation PDT efficacy.

Multifunctional Phosphorescent Conjugated Polymer Dots for Hypoxia Imaging and Photodynamic Therapy of Cancer Cells.

Molecular oxygen (O2) plays a key role in many physiological processes, and becomes a toxicant to kill cells when excited to 1O2. Intracellular O2 levels, or the degree of hypoxia, are always viewed as an indicator of cancers. Due to the highly efficient cancer therapy ability and low side effect, photodynamic therapy (PDT) becomes one of the most promising treatments for cancers. Herein, an early‐stage diagnosis and therapy system is reported based on the phosphorescent conjugated polymer dots (Pdots) containing Pt(II) porphyrin as an oxygen‐responsive phosphorescent group and 1O2 photosensitizer. Intracellular hypoxia detection has been investigated. Results show that cells treated with Pdots display longer lifetimes under hypoxic conditions, and time‐resolved luminescence images exhibit a higher signal‐to‐noise ratio after gating off the short‐lived background fluorescence. Quantification of O2 is realized by the ratiometric emission intensity of phosphorescence/fluorescence and the lifetime of phosphorescence. Additionally, the PDT efficiency of Pdots is estimated by flow cytometry, MTT cell viability assay, and in situ imaging of PDT induced cell death. Interestingly, Pdots exhibit a high PDT efficiency and would be promising in clinical applications.

Photodynamic therapy induced cell death of hormone insensitive prostate cancer PC-3 cells with autophagic characteristics

The introduction of photodynamic therapy (PDT) to the treatment of advanced prostate cancer can accomplish the eradication of local neoplasm and distant metastases with minimized damage to the adjacent structures. The evidence of PDT efficacy for androgen-refractory prostate cancer will be especially meaningful for the patients resistant to hormone therapy. Pheophorbide a (PhA) as a photosensitizer was employed to evaluate the photodynamic efficacy in androgen-insensitive PC-3 prostate cancer cells in culture by cell viability assay, reactive oxygen species (ROS) measurement and cell cycle test. Characteristics of apoptosis and autophagy were investigated via DNA fragmentation electrophoresis and immune-fluorescence staining, acidic vesicle determination and detection of LC3B in puncta form by fluorescence microscopy, Western blotting of autophagy-related (Atg) proteins and detailed phenotype shown by electron microscopy. PhA exerted significant photo-cytotoxicity toward androgen-insensitive prostate cancer PC-3 cells in photosensitizer-dose and light-dose dependent manners. The photoactivation immediately initiated hyperproduction of ROS, the depolarization of mitochondrial membrane potential and the arrest of the cell cycle in the G0/G1 phase. Autophagy was revealed in PhA-PDT treated PC-3 cells by a significant high amount of acidic vesicular organelles with acridine orange staining, recruitment of LC3B on the membrane of autophagosomes by fluorescent microscopy, double membrane-bound vesicles suggesting autophagosomes by electron microscopy, significant increased Atg proteins such as beclin-1, Atg12-Atg5 conjugation, Atg7 and the conversion of LC3B-I to LC3B-II by Western blot analysis. PhA-mediated PDT induced significant autophagy in hormone-refractory prostate cancer PC-3 cells.

Down‐regulation of heat‐shock protein 27–induced resistance to photodynamic therapy in oral cancer cells

Photodynamic therapy (PDT) of cells is a new treatment modality involving selective delivery of a photosensitive dye into target cells, followed by visible light irradiation. PDT induces cell death by excessive ROS generation. The effects of multiple photosensitizers were owing to the difference in cell types involving sensitizer-specific protein changes linked to resistance. HSP27 is regulated in response to stress and is associated with apoptotic process. The effects of HSP27 on PDT resistance are controversial and unclear. The purpose of this study was to investigate the role of HSP27 down-regulation in the PDT-induced cells and HSP27 regulation in the resistance to PDT. KB cells transfected with HSP27 siRNA were exposed to hematoporphyrin (HP) followed by irradiation at 635 nm at an energy density of 4.5 mW/cm(2). After irradiation, the effects on HSP27 down-regulation were assessed by MTT assay, flow cytometry, confocal analysis, Western blotting and caspase activity. The results of this study showed that down-regulation of HSP27 restored cell survival in HP-PDT-induced apoptotic KB cells. HSP27 down-regulation attenuated PDT-induced apoptosis through caspase-mediated pathway in KB cells. Also, HSP27 silencing regulated Bax, Bcl-2, and PARP protein expression in PDT-induced cells. Therefore, HSP27 down-regulation confers resistance to PDT through interruption of apoptotic protein activity in PDT-induced cell death. HSP27 might contribute to regulating PDT-induced apoptosis in PDT-resistant cells.

Abstract 3464: Aminolevulinic acid-mediated photodynamic therapy induces cell death in human osteosarcoma cells

Abstract Photodynamic therapy (PDT) is a localized cancer treatment that requires a photosensitizer (PS), molecular oxygen, and light to induce cell death. Aminolevulinic acid (ALA)-mediated PDT utilizes ALA as an inert precursor to the required PS. Subsequent to cellular uptake ALA is converted to a PS, protoporphyrin IX (PpIX), in the mitochondria via the heme pathway. The excitation of PpIX by light can then result in the generation of reactive oxygen species, leading to direct tumor cell death, damage to the microvasculature, and induction of local inflammatory response. Interestingly, PDT can evoke apoptotic and/or necrotic cell death pathways within the same cell population. The balance between apoptosis and necrosis depends on several parameters, including the total PDT dose, the intracellular localization of the PS, the fluence rate, and the cell type. Recent studies show that the cell death mechanism induced has a direct influence on the systemic immune response. Therefore, a better understanding of the interplay between different cell death mechanisms in response to PDT could lead to improved tumor response and could inform better therapeutic strategies. We first determined the cytotoxicity of ALA to the human osteosarcoma cell line MG-63. PDT is a relatively unexplored method of treatment for human osteosarcoma, and could represent a superior method to existing therapies. Data from our lab show that ALA is not significantly cytotoxic to MG-63 cells, and that it is efficiently converted to PpIX within 24 hours of treatment. We also demonstrate that ALA-mediated PDT significantly induces cell death in MG-63 cells, and that cell death increases with increased ALA concentration and light fluence. Our findings indicate that this cell death results from a combination of both apoptosis and necrosis, with necrosis representing the major pathway. Finally, we show that increased light fluence decreases the amount of observed apoptosis subsequent to ALA-mediated PDT. These results taken together suggest that ALA-mediated PDT can induce cell death in human osteosarcoma cells, and that most cells are dying by necrosis. Furthermore, our data suggests that by adjusting specific therapeutic parameters, we can shift the balance between separate death pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3464. doi:1538-7445.AM2012-3464

Vitamin D pretreatment augments aminolevulinate photodynamic therapy-induced cell death by upregulation of tumor necrosis factor alpha and caspase-8 in mouse models of skin carcinoma

Vitamin D pretreatment augments aminolevulinate photodynamic therapy-induced cell death by upregulation of tumor necrosis factor alpha and caspase-8 in mouse models of skin carcinoma

Zinc phthalocyanines-mediated photodynamic therapy induces cell death in adenocarcinoma cells

A panel of eight Zn-phthalocyanines (Zn-Pcs) ( 1)–( 8), differently substituted on the benzo units, was synthesized either by direct cyclic-tetramerization of substituted phthalonitriles (compounds 1 and 2), or leading from the easily available tetrasulphonyl phthalocyanine to yield the sulfonamido derivatives 3 and 4, or else via the chloromethylation of precedent Zn-Pc followed by reaction with nucleophiles affording the dicationic Zn-Pcs ( 5) and ( 6) or the neutral Zn-Pcs ( 7) and ( 8). The phototoxicity of these new compounds was evaluated in vitro on human colon adenocarcinoma cell line (HCT116), and their effect compared with those induced by porfimer sodium. The results are reported as IC 50 values, following exposure of the cells to different Zn-Pcs concentration and irradiation with a 500 W tungsten/halogen white lamp. The cationic Zn-Pc ( 5) and ( 6) together with the Zn-Pc ( 7), featuring 12 methoxy groups, were found good or fairly good photosensitizers while the more lipophilic Zn-Pcs ( 1)–( 4) and ( 8) were found devoid of activity.

Phthalocyanine-mediated photodynamic therapy induces cell death and a G 0/G 1 cell cycle arrest in cervical cancer cells

We have developed a series of novel photosensitizers which have potential for anticancer photodynamic therapy (PDT). Photosensitizers include zinc phthalocyanine tetra-sulphonic acid and a family of derivatives with amino acid substituents of varying alkyl chain length and degree of branching. Subcellular localization of these photosensitizers at the phototoxic IC 50 concentration in human cervical carcinoma cells (SiHa Cells) was similar to that of the lysosomal dye Lucifer Yellow. Subsequent nuclear relocalization was observed following irradiation with 665 nm laser light. The PDT response was characterized using the Sulforhodamine B cytotoxicity assay. Flow cytometry was used for both DNA cell cycle and dual Annexin V-FITC/propidium iodide analysis. Phototoxicity of the derivatives was of the same order of magnitude as for tetrasulphonated phthalocyanine but with an overall trend of increased phototoxicity with increasing amino acid chain length. Our results demonstrate cell death, inhibition of cell growth, and G 0/G 1 cell cycle arrest during the phthalocyanine PDT-mediated response.

Photodynamic Therapy induced Cell Death using ALA and 632nm Diode Laser in A549 Lung Cancer Cells

Background : Photodynamic therapy (PDT) is a new therapeutic method aimed at the selective destruction of cancer cells. The outcome is death of cancer cells through apoptosis or necrosis. The aim of this study was to investigate the characterization of PDT induced cell death in A549 lung cancer cells. Materials and methods : A549 cells were used as the lung cancer cell. 5 aminolevulinic acid (ALA) was used as the photosensitizer and a 632nm diode laser (Biolitec, Germany) as the light source. Cells were incubated with various concentrations of ALA. The 632nm diode laser was then administered for various laser irradiation times. The treated cells were incubated with 24, 48 and 72 hours. The cell viabilities were measured using the crystal violet assay and light microscopy. To observe the cell death mechanism after PDT, cells were observed under fluorescence microscopy after double staining with Hoechst 33342 and propium iodide after PDT. Results : In the crystal violet assay at 24 hours after PDT with a laser irradiation power, the cell viabilities were , , and with ALA concentrations of 10, 100, and , respectively. In crystal violet assay at 24 hours after PDT with of ALA, the cell viabilities were , , and with 1.6, 3.2 and laser irradiation powers, respectively. However, increasing the interval time after PDT did not change the cell viabilities. In the apoptosis assay, photodynamic therapy was inducing the apoptotic cell death. Conclusions : This study shows the apoptotic anticancer effect of photodynamic therapy in A549 lung cancer cells. However, further evaluations with other cancer cells and photosensitizers are necessary.

PHOTODYNAMIC THERAPY WITH PHOTOFRIN II INDUCES PROGRAMMED CELL DEATH IN CARCINOMA CELL LINES

The mode of cell death following photodynamic therapy was investigated from the perspective of programmed cell death or apoptosis. Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a) and rat mammary carcinoma (MTF7) were treated by photodynamic therapy. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder indicative of internucleosomal cleavage of DNA during apoptosis. The magnitude of the response and the photodynamic therapy dosage required to induce DNA fragmentation were different in PC3 and MTF7. The MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal response at the LD50 but had a marked response at the LD85. Thus, apoptosis did not ensue as quickly in PC3 as in MTF7. The H322a cells were killed by photodynamic therapy but failed to exhibit any apoptotic response. The results also suggested that apoptosis in these cell lines has a minor requirement for de novo protein synthesis and no requirement for de novo RNA synthesis. This study indicates that although apoptosis can occur during photodynamic therapy-induced cell death, this response is not universal for all cancer cell lines.