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。     

Photofrin--factor of photodynamic therapy induces apoptosis and necrosis

Photodynamic therapy (PDT) causes irreversible photodamage of tumor and other malignant tissues. The effect of reactive oxygen species generation in the presence of photofrin (HpD) was studied. The studies were performed on endothelial cell line from foetal aorta of calves and on normal fibroblasts cell line (3T3 -Balb) and also on malignant line (A431). The cells were grown in presence of photofrin at different time intervals. Time of interaction of photosensitiser with cells was very important. Short time of exposure of the cells to photofrin induced mostly apoptosis in normal cells and apoptotic or necrotic changes in malignant cells. Longer effect of these factors on cells provoked necrosis. The factors of PDT influence dynamic changes of SOD and CT activity. It was dependent on the intensity of factors. These results strongly suggest that HpD has an effect on generation of ROS, which are a signal for development of morphological changes (apoptosis or necrosis) in normal and malignant cells.     

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.     

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.     

Methylene blue-mediated photodynamic therapy induces mitochondria-dependent apoptosis in HeLa cell

Methylene blue (MB), a widely studied reagent, is investigated in this work for its usage in photodynamic therapy (PDT). PDT has been proved to be highly effective in the treatment of different types of cancers. Previous studies showed MB has both high affinity for mitochondria and high photodynamic efficiency. To elucidate the effects of MB in PDT, we analyzed PDT-induced apoptosis in HeLa cells by introducing different doses of MB into the culture media. Our data showed that MB-mediated PDT triggered intense apoptotic cell death through a series of steps, beginning with photochemical generation of reactive oxygen species. The release of cytochrome c and activation of caspase-3 indicated that MB-PDT-mediated apoptosis in HeLa cells was executed by the mitochondria-dependent apoptotic pathway. Importantly, proteomic studies confirmed that expression levels of several mitochondrial proteins were altered in MB-PDT-induced apoptosis, including TRAP1, mitochondrial elongation factor Tu and peroxiredoxin 3 isoform b. Western blot data showed that phosphorylation of ERK1/2 and PKA were reduced in MB-PDT treated cells, indicating several signal molecules participating in this apoptotic cascade. Moreover, MB-PDT induced an increase in the strength of interaction between Bcl-xL and dephosphorylated Bad. This led to loss of the pro-survival function of Bcl-xL and resulted in mitochondria-mediated apoptosis. This study provides solid evidence of a strong induction by MB-PDT of a mitochondria-dependent apoptosis cascade in HeLa cells.     

New directions in photodynamic therapy.

Photodynamic therapy (PDT), a treatment approach that makes use of a photosensitizer to generate a localized toxic species in diseased tissue, has recently become an approved treatment modality. So far, however, only a handful of photosensitizers have received regulatory approval and for a small number of diseases. This chapter outlines the major limitations of PDT and speculates on the possible improvements that are required in order to advance PDT to a front line therapy. Seven areas of improvements are discussed: drug selectivity, drug delivery, light delivery, combination therapies, pigmented tumors, other potential uses, and protocol optimization. For each area, current limitations are discussed, and further required studies are recommended.     

Antibacterial photodynamic therapy in dermatology.

Photodynamic therapy (PDT) appears to be endowed with several favourable features for the treatment of localized microbial infections, especially after the advent of cationic photosensitising agents (phenothiazines, meso-substituted porphyrins, polylysine-bound chlorins) which properly interact with the outer wall at the surface of several types of bacterial and yeast cells, increase their permeability, and allow significant amounts of photosensitizer to be accumulated at the level of the cytoplasmic membrane. These photosensitisers are characterized by a broad spectrum of activity, being effective toward both wild strain and antibiotic-resistant gram-positive and gram-negative bacteria and yeasts. In general, extensive eradication of pathogens can be achieved under mild irradiation conditions, such as short incubation times and low fluence-rates, which guarantees a high degree of selectivity in comparison with the main constituents of host tissues, such as keratinocytes and fibroblasts. Moreover, the photosensitised inactivation of microorganisms is typically a multi-target process; as a consequence, the selection of photoresistant microbial strains is very unlikely and has not been experimentally observed so far. Possible initial targets of antimicrobial PDT applications include periodontal diseases, impetigo, atopic dermatitis, acne vulgaris, infected wounds, and superinfected posriatic plaques.     

Molecular effectors of multiple cell death pathways initiated by photodynamic therapy

Photodynamic therapy (PDT) is a recently developed anticancer modality utilizing the generation of singlet oxygen and other reactive oxygen species, through visible light irradiation of a photosensitive dye accumulated in the cancerous tissue. Multiple signaling cascades are concomitantly activated in cancer cells exposed to the photodynamic stress and depending on the subcellular localization of the damaging ROS, these signals are transduced into adaptive or cell death responses. Recent evidence indicates that PDT can kill cancer cells directly by the efficient induction of apoptotic as well as non-apoptotic cell death pathways. The identification of the molecular effectors regulating the cross-talk between apoptosis and other major cell death subroutines (e.g. necrosis, autophagic cell death) is an area of intense research in cancer therapy. Signaling molecules modulating the induction of different cell death pathways can become useful targets to induce or increase photokilling in cancer cells harboring defects in apoptotic pathways, which is a crucial step in carcinogenesis and therapy resistance. This review highlights recent developments aimed at deciphering the molecular interplay between cell death pathways as well as their possible therapeutic exploitation in photosensitized cells.     

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.     

Multiple pathways to apoptosis.

Evidence from studies dealing with various aspects of the interdigitating network of proliferative signals supports the concept of multiple signalling pathways. Analogous to these, the concept of multiple pathways to apoptosis is introduced and discussed. Some of these pathways might not necessarily be under genetic control. They might be elicited by a perturbation at any point in the proliferative signalling network, for example by an imbalance in any of the substances involved directly or as second messengers. Diverse findings from recent reports on the induction of apoptosis by various means and in a variety of cell lines imply the presence of such multiple pathways, only one of which should accurately be described as genetic.     

Signaling pathways in apoptosis as potential targets for cancer therapy

Genetic instability contributes to the origin of cancer as well as to the ability of cancer cells to become resistant to various therapies. Because of this, cytotoxic rather than cytostatic therapies might be most effective against this disease. Many oncogenes and tumor suppressors mediate their effects by interfering with or inducing apoptotic signaling. Thus, apoptotic pathways might be significantly altered in cancer cells relative to untransformed cells, and these differences might present a therapeutic window that can be exploited for development of cancer drugs.     

Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy

Rose Bengal acetate photodynamic therapy (RBAc–PDT) induced multiple cell death pathways in HeLa cells through ROS and ER stress. Indeed, apoptosis was the first preferred mechanism of death, and it was triggered by at least four different pathways, whose independent temporal activation ensures cell killing when one or several of the pathways are inactivated. Apoptosis occurred as early as 1 h after PDT through activation of intrinsic pathways, followed by activation of extrinsic, caspase-12-dependent and caspase-independent pathways, and by autophagy. The onset of the different apoptotic pathways and autophagy, that in our system had a pro-death role, was timed by determining the levels of caspases 9, 8, 3 and 12; Bcl-2 family; Hsp70; LC3B; GRP78 and phospho-eIF2α proteins. Interestingly, inhibition of one pathway, that is, caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting that the independent onset of the different apoptotic pathways and autophagy did not occur in a subordinated manner. Altogether, our data indicate RBAc as a powerful photosensitiser that induces a prolonged cytotoxicity and time-related cell death onset by signals originating from or converging on almost all intracellular organelles. The fact that cancer cells can die through different mechanisms is a relevant clue in the choice and design of anticancer PDT.     

Regulatory pathways in the European Union

In principle, there are three defined procedures to obtain approval for a medicinal product in the European Union. As discussed in this overview of the procedures, the decision on which regulatory pathway to use will depend on the nature of the active substance, the target indication(s), the history of product and/or the marketing strategy.     

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.     

Promotion of photodynamic therapy-induced apoptosis by stress kinases.

Photodynamic therapy (PDT), a cancer treatment that employs a photosensitizer and visible light, induces apoptosis in murine LY-R leukemic lymphoblasts and in CHO cells, but the rate and extent of apoptosis are much greater in LY-R cells. Three MAPK family members, ERK1/ERK2, SAPK/JNK, and p38/HOG, are important intermediates in signal transduction pathways. To ascertain whether activation of one or more MAPKs could mediate PDT-induced apoptosis, Western blot analysis has been performed on the proteins of LY-R and CHO cells at various times following lethal (90 - 99% cell kill) doses of PDT photosensitized by the phthalocyanine Pc 4. The blots were probed with antibodies to each of the proteins as well as antibodies specific for the activated (phosphorylated) forms of each kinase. Of the three MAPK types, only the p46 and p54 SAPK/JNKs were found to be activated by PDT in LY-R cells, with a maximum approximately threefold increase in the content of the phosphorylated forms reached in 30 - 60 min. An even larger relative activation was observed in CHO cells. PDT did not affect ERK and p38/HOG activation in LY-R cells. In the case of CHO cells, however, ERK2 was slightly activated at 5 min post-PDT, then declined, and p38/HOG was strongly activated from 5 to 60 min post-PDT. A specific inhibitor (PD98059) of MEK1, the kinase that activates ERK, had little or no effect on PDT-induced apoptosis in either LY-R or CHO cells. In contrast, a specific inhibitor of p38/HOG (SB202190) blocked PDT-induced apoptosis in LY-R cells with a lesser effect in CHO cells. The results suggest that both the SAPK and p38/HOG cascades can be stimulated by PDT and that the latter participates in both rapid and slow PDT-induced apoptosis. Furthermore, the high level of constitutively active p38/HOG in LY-R cells may poise those cells for rapid activation of apoptosis following PDT.     

Erythrocytes-the 'house elves' of photodynamic therapy.

Photodynamic therapy (PDT) and fluorescence diagnosis (FD) are being developed for a number of clinical applications. Since fluorophores and photosensitising drugs are usually given systemically their effect on blood elements are of significant importance. Photodynamic effects on erythrocytes occur naturally in patients with erythropoietic protoporphyria (EPP). Exposure to small fluences, as obtained by the erythrocytes when they pass capillaries in the skin, leads to transfer of the photosensitiser protoporphyrin IX (PP IX), from EPP erythrocytes to endothelial cells. Thus, the erythrocytes are partly protected while the endothelial cells suffer photodamage. During photodynamic therapy in vivo erythrocytes are regularly photosensitised. This side effect is partly intended but mostly unwanted, and a summary of this topic is given. Furthermore, the effect of UV-A on erythrocytes that is accompanied with the formation of bilirubin is reviewed. Erythrocytes serve as convenient model cells for experimental research. Such use of erythrocytes to screen new photosensitisers may be of limited value. A combination of photohaemolysis and haemoglobin oxygenation may become the basis for an assay for in vitro phototoxicity. Erythrocytes from birds are good model cells for exploration of physiological and molecular mechanisms involved in PDT. A potential mechanism of PDT induced behaviour resembling apoptosis in erythrocytes is provided.PDT for sterilisation of erythrocyte concentrates has a potential for medical use. Photodynamic effects on the erythrocytes themselves should be avoided. This is realised by choosing a virus-selective photosensitiser, low fluences and treatment of the concentrates with agents like dipyridamole and antioxidants. Future aspects of applications of photosensitisation of red blood cells are discussed.     

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).     

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.