Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.     

ZnPcS2P2-based photodynamic therapy induces mitochondria-dependent apoptosis in K562 cells

Mitochondria play a key role in the regulation of apoptosis induced by numerous antitumor chemotherapeutic and other toxic agents. Photodynamic therapy (PDT) exerts significant cellular killing efficacy through either an apoptotic or necrotic cell death pathway. This study investigated the mechanism underlying the killing effects of a novel amphipathic photosensitizer [di-sulfonated di-phthalimidomethy lphthalocyanine zinc (ZnPcS2P2)]-mediated photodynamic therapy (ZnPcS2P2-PDT) on K562 cells. Apoptosis was evident in the post-PDT cells through the TdT-mediated dUTP nick end labeling (TUNEL) method and DNA fragmentation assay. After ZnPcS2P2-PDT, K562 cells underwent mitochondria-dependent apoptosis as evidenced by the release of cytochrome c from mitochondria into cytosol, accompanied by mitochondrial membrane potential (A~m) reduction, indicating the opening of the mitochondrial permeability transition pore (PTP). The activities of protease from the caspase family and caspase-3 were also significantly elevated. Furthermore, ZnPcS2P2-PDT down-regulated the expression of chimaeric Bcr-Abl oncoprotein, which is the molecular hallmark of chronic myelogenous leukemia (CML).     

Initiation of apoptosis and autophagy by photodynamic therapy

This study was designed to examine modes of cell death after photodynamic therapy (PDT). Murine leukemia L1210 cells and human prostate Bax-deficient DU-145 cells were examined after PDT-induced photodamage to the endoplasmic reticulum (ER). Previous studies indicated that this treatment resulted in a substantial loss of Bcl-2 function. Both apoptosis and autophagy occurred in L1210 cells after ER photodamage with the latter predominating after 24 hr. These processes were characterized by altered cellular morphology, chromatin condensation, loss of mitochondrial membrane potential and formation of vacuoles containing cytosolic components. Western blots demonstrated processing of LC3-I to LC3-II, a marker for autophagy. In DU145 cells, PDT initiated only autophagy. Phosphatidylinositol (PI) 3-kinase inhibitors suppressed autophagy in both cell lines as indicated by inhibition of vacuolization and LC3 processing. Inhibitors of apoptosis and/or autophagy were then used to delineate the contributions of the two pathways to the effects of PDT. Given the ability of autophagy to upregulate MHC-11 peptide presentation, autophagy may play a role in the ability of photodynamic therapy to stimulate immunologic recognition of target cells.     

Combination of photodynamic therapy with aspirin in human-derived lung adenocarcinoma cells affects proteasome activity and induces apoptosis

Objectives: Photodynamic treatment (PDT) of human lung carcinoma cells A549 (p53^+/+) and H1299 (p53^?/?) induces fast but transient stalling of proteasome activity. We have explored the possibility of prolonging this effect by combining PDT with drugs capable of sustaining the stall, and promote apoptosis of surviving cells. We show that aspirin can be used to accomplish this. Materials and methods: Cells were irradiated at doses ranging from 0.54 to 1.10 J cm^?2, and subsequently were incubated with aspirin at either high (10 and 5 mm ) or low concentration (2.5 and 1.5 mm ). Photofrin concentration and incubation time were constant (2.5 μg/ml and 16 h). Under these conditions, we analysed cell viability, colony‐forming efficiency, cycle profile, expression patterns of specific proteins and ubiquitination state, after individual or combined administration. Results: Treatment with either PDT or aspirin, rapidly induced proteasome malfunction and accumulation of cells in G₂M, but did not induce apoptosis. However, when aspirin was added to cells (even at low concentrations) after PDT, the proteasome block was sustained. Moreover, significant cytotoxic effects, including apoptosis, were observed along with cytostatic effects (G₂M accumulation/decreased colony formation). Conclusions: Combination of PDT and low‐toxicity drugs (such as aspirin) resulted in protracted inhibition of proteasome activity and induced apoptosis even in apoptosis‐resistant cancer cells.     

Regulatory pathways in photodynamic therapy induced apoptosis.

Photodynamic therapy is an approved treatment for several types of tumors and certain benign diseases, based on the use of a light-absorbing compound (photosensitizer) and light irradiation. In the presence of molecular oxygen, light-activation of the photosensitizer, which accumulates in cancer tissues, leads to the local production of reactive oxygen species that kill the tumor cells. Mitochondria are central coordinators of the mechanisms by which PDT induces apoptosis in the target cells. Recent studies indicate that concomitant to the permeabilization of the outer mitochondrial membrane (which leads to the release of several apoptogenic factors in the cytosol and to the activation of effector caspases), regulatory signaling pathways are activated in a photosensitizer, PDT dose and cell-dependent fashion. Signaling pathways regulated by members of mitogen activated protein kinases and their downstream targets, such as cyclooxygenase-2, appear to critically modulate cancer cell sensitivity to PDT. Understanding the molecular events that contribute to PDT-induced apoptosis, and how cancer cells can evade apoptotic death, should enable a more rationale approach to drug design and therapy.     

Triple therapy with octreotide,galanin and serotonin induces necrosis and increases apoptosis of a rat colon carcinoma

A rat colonic adenocarcinoma was implanted subcutaneously (s.c.) in nude mice. After 7 days, the animals were divided into different groups. Two groups received subcutaneous injections twice daily with 3 or 6 micro g/kg body weight octreotide, galanin and serotonin. Three groups were respectively treated with 20, 30, and 40 micro g/kg body weight of the previously mentioned bioactive substances. Control group received only saline solution in the same fashion as treated animals. The treatment lasted for 5 days. The tumour volume and weight, the relative density of blood vessels, of tumour necrotic tissue, of apoptotic nuclei and of proliferating nuclei were measured. Apoptosis was detected by in situ labelling of nuclear DNA fragmentation according to TUNEL method, and proliferation by immunocytochemistry. Morphometry was done with the classical stereological point-counting method. Food consumption, animal weight, faeces weight and its water content were measured for 3 days before and after treatment. Triple therapy with 3 and 6 micro g/kg body weight had no effect on any of the parameters measured, except in reducing the relative volume density of tumour blood vessels. Treatment with 20, 30 and 40 micro g/kg body weight of the previously mentioned bioactive substances reduced the tumour volume, the relative volume density of blood vessels and increased the relative volume density of necrotic tissue and of apoptotic nuclei (in the 20 micro g group). However, there was no difference between treated mice and controls regarding the relative volume density of proliferating nuclei. There was no statistical difference between treated animals regarding food consumption, body weight, faeces weight and its water content before and during treatment. The present study confirms that triple therapy with octreotide, galanin and serotonin causes regression of a rat colon carcinoma. It further showed that optimum treatment dose is 20 micro g/kg body weight of each bioactive substance. Moreover, this therapy regime does not show apparent side effects in the experiments carried out on mice.     

Tumor necrosis factor-alpha induces apoptosis in rat brown adipocytes

Accumulating evidence demonstrates that adipose tissue is a major site of tumor necrosis factor-alpha (TNF-alpha) gene expression, which is markedly high in obese animals and may contribute to obesity-linked insulin resistance. We now report that recombinant murine TNF-alpha triggers the apoptotic degeneration of brown adipocytes differentiated in culture. Moreover, noradrenaline, which has been described as having trophic effects on brown fat and accelerating the differentiation of brown adipocytes, is capable of dose-dependently preventing the TNF-alpha-induced apoptosis of brown fat cells. Since obesity is characterized by greatly increased TNF-alpha production and reduced catecholaminergic activity, apoptosis was studied in the brown fat of genetically obese animals. In situ DNA fragmentation analysis revealed a larger number of apoptotic cells in the brown fat of obese (fa/fa) than in that of lean (+/+) Zucker rats. The exposure of obese rats to low temperatures for 7 days, which increases the sympathetic activity of brown adipose tissue, significantly reduces the number of apoptotic brown adipocytes. We hypothesize that TNF-alpha may play a significant role in the control of brown fat homeostasis.     

Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis

Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.     

The influence of photodynamic therapy on apoptosis in human melanoma cell line

Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegr?m Melanoma (BM) cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 microg/ml and 30 microg/ml) 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm). The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 microg/ml and 30 microg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.     

Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin

Surface-exposed HSP70 and calreticulin are damage-associated molecular patterns (DAMPs) crucially involved in modulating the success of cancer therapy. Photodynamic therapy (PDT) involves the administration of a photosensitising (PTS) agent followed by visible light-irradiation. The reactive oxygen species that are thus generated directly kill tumours by damaging their microvasculature and inducing a local inflammatory reaction. PDT with the PTS photofrin is associated with DAMPs exposure, but the same is not true for other PTSs. Here, we show that when cancer cells are treated with hypericin-based PDT (Hyp-PDT), they surface-expose both HSP70 and calreticulin (CRT). Induction of CRT exposure was not accompanied by co-exposure of ERp57, but this did not compromise the ability of the exposed CRT to regulate the phagocytosis of Hyp-PDT-treated cancer cells by dendritic cells. Interestingly, we found that Hyp-PDT-induced CRT exposure (in contrast to anthracycline-induced CRT exposure) was independent of the presence of ERp57. Our results indicate that Hyp-PDT is a potential anti-cancer immunogenic modality.     

Inflammation of choriodecidua induces tumor necrosis factor alpha-mediated apoptosis of human myometrial cells

The present study investigated the ability of human choriodecidua to induce myometrial cell apoptosis through the secretion of tumor necrosis factor alpha (TNF). The secretion of TNF was evaluated in the culture supernatants of amnion and choriodecidua explants that were exposed to the bacterial endotoxin lipopolysaccharide (LPS) to mimic inflammation. The choriodecidua explants produced more TNF than the amnion explants in response to LPS stimulation, despite the fact that the choriodecidua had lower levels of TLR4 expression. Moreover, conditioned medium obtained from LPS-treated choriodecidua explants, but not that from amnion explants, decreased the number of viable cultured myometrial cells and induced cell apoptosis by inducing the overexpression of the proapoptotic protein BAX and by decreasing the expression of the anti-apoptotic protein BCL2. Neutralization of TNF in the choriodecidua-conditioned medium reversed this effect. Exogenous TNF mimicked LPS-treated choriodecidua-conditioned medium in that it induced myometrial cell apoptosis, reduced BCL2 expression, and increased BAX expression. Using neutralizing antibodies against both subtypes of TNF receptors, we found that only TNFRSF1A participates in TNF-induced myometrial cell apoptosis. Our in vitro model of LPS-induced inflammation of human fetal membrane explants suggests a mechanism by which TNF secreted by choriodecidua governs human myometrial cell apoptosis at the end of pregnancy. These data support the hypothesis that TNF participates in the complex network of signaling processes associated with uterine involution.     

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

Background

The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.

Methods

Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.

Results

m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.

Conclusions

Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.

General significance

These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.     

Antifungal photodynamic therapy

In photodynamic antimicrobial chemotherapy (PACT), a combination of a sensitising drug and visible light causes selective destruction of microbial cells. The ability of light-drug combinations to kill microorganisms has been known for over 100 years. However, it is only recently with the beginning of the search for alternative treatments for antibiotic-resistant pathogens that the phenomenon has been investigated in detail. Numerous studies have shown PACT to be highly effective in the in vitro destruction of viruses and protozoa, as well as Gram-positive and Gram-negative bacteria and fungi. Results of experimental investigations have demonstrated conclusively that both dermatomycetes and yeasts can be effectively killed by photodynamic action employing phenothiazinium, porphyrin and phthalocyanine photosensitisers. Importantly, considerable selectivity for fungi over human cells has been demonstrated, no reports of fungal resistance exist and the treatment is not associated with genotoxic or mutagenic effects to fungi or human cells. In spite of the success of cell culture investigations, only a very small number of in vivo animal and human trials have been published. The present paper reviews the studies published to date on antifungal applications of PACT and aims to raise awareness of this area of research, which has the potential to make a significant impact in future treatment of fungal infections.     

Properties and applications of photodynamic therapy

Photodynamic therapy (PDT) is the treatment of malignant lesions with visible light following the systemic administration of a tumor-localizing photosensitizer. Pharmacological and photochemical properties of the photosensitizer are combined with precise delivery of laser-generated light to produce a treatment which can offer selective tumoricidal action. Hematoporphyrin derivative (HD) and a purified component called Photofrin II are currently being used in clinical PDT. Initial patient results have been encouraging, and considerable interest has developed in the synthesis and evaluation of new photosensitizers with improved photochemical and pharmacological characteristics. In addition, there has been a gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT. This report provides an overview of the properties and applications of PDT. Information and data related to drug development, photochemistry, subcellular targets, in vivo responses, and clinical trials of PDT are presented.     

Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

Background

Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential.

Results

The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude.

Conclusions

Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.     

Zinc pyrithione induces ERK- and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells

Zinc dyshomeostasis can induce cell death. However, the mechanisms involved have not been fully elucidated in prostate cancer (PCa) cells, which differ dramatically from normal cells in their zinc handling ability. Here, we studied the effects of the ionophore Zn-pyrithione (ZP) and the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Both compounds induced cell death at micromolar concentrations when incubated with androgen-dependent (LNCaP), androgen-independent (PC3, DU145) and androgen-sensitive (C4-2) PCa cell-lines. Compared to PCa cells, RWPE1 prostate epithelial cells were less sensitive to ZP and more sensitive to TPEN, but total cellular zinc levels were changed similarly. ZnSO4 enhanced the toxicity of ZP, but inhibited the effects of TPEN as expected. The morphological/biochemical responses to ZP and TPEN differed. ZP decreased ATP levels and stimulated ERK, AKT and PKC phosphorylation. DNA laddering was observed only at low doses of ZP but all doses of TPEN. TPEN activated caspase 3/7 and induced PARP-cleavage, DNA-fragmentation, ROS-formation and apoptotic bodies. PKC and ERK-pathway inhibitors, and antioxidants protected against ZP-induced but not TPEN-induced death. Inhibitors of MPTP-opening protected both. Cell death in response to TPEN (but not ZP) was diminished by a calpain inhibitor and largely prevented by a caspase 3 inhibitor. Overall, the results indicated primarily a necrotic cell death for ZP and an apoptotic cell death for TPEN. The enhanced sensitivity of PCa cells to ZP and the apparent ability of ZP and TPEN to kill quiescent and rapidly dividing cells in a p53-independent manner suggest that ZP/TPEN might be used to develop adjunct treatments for PCa.     

Stem cell-based photodynamic therapy

We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.     

肿瘤光动力疗法诱导细胞凋亡机制研究进展

肿瘤光动力疗法（Photodynamic Therapy,PDT）是利用光敏剂分子接受光照后产生多种活性氧物质（reactive oxyger,ROS),使细胞结构和功能受到损伤,而导致细胞凋亡的一种独特的肿瘤治疗方法,已受到越来越多的重视。本文对近几年有关PDT诱导肿瘤细胞凋亡方面的研究进展作了综述性介绍。