Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells

Photodynamic therapy (PDT) is a novel cancer treatment utilizing a photosensitizer, visible light and oxygen. PDT with the silicon phthalocyanine Pc 4, a new photosensitizer, is highly effective in cancer cell destruction and tumor ablation. The mechanisms underlying cancer cell killing by PDT are not fully understood. Tumor necrosis factor alpha (TNF) is a multifunctional cytokine that has been implicated in photocytotoxicity. We asked whether recombinant human TNF (rhTNF) affects Pc 4-PDT cytotoxicity in A431 human epidermoid carcinoma cells. Co-treatment of A431 cells with various doses of Pc 4-PDT and a sub-lethal rhTNF dose led to a sub-additive reduction in cell survival. In addition, in the presence of Pc 4-PDT or rhTNF, caspase-3 activity and apoptosis were induced. The combined treatment, however, did not potentiate either caspase-3 activity or apoptosis. Similar to previous findings we observed that Pc 4-PDT initiated a time-dependent extracellular TNF accumulation. The data suggest that: a) PDT and rhTNF induce cancer cell killing through different mechanisms; and b) Pc 4-PDT-induced TNF production is a stress response that may not directly affect photocytotoxicity.     

Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 of SW480 human colon cancer xenografts in athymic mice.

Photodynamic therapy (PDT) using the silicon phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N-(CH3)2] is an oxidative stress associated with induction of apoptosis in various cell types. We assessed the effectiveness of Pc 4-PDT on SW480 colon cancer xenografts grown in athymic nude mice. Animals bearing xenografts were treated with 1 mg/kg body weight Pc 4 and 48 h later were irradiated with 150 J/cm2 672-nm light from a diode laser delivered at 150 mW/cm2. Biochemical studies were performed in xenografts resected at various time points up to 26 h after Pc 4-PDT treatment, whereas tumor size was evaluated over a 4-week period in parallel experiments. In the tumors resected for biochemical studies, apoptosis was visualized by activation of caspase-9 and caspase-3 and a gradual increase in the cleavage of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) to a maximum of approximately 60% of the total PARP present at approximately 26 h. At that time all Pc 4-PDT-treated tumors had regressed significantly. Two signaling responses that have previously been shown to be associated with Pc 4-PDT-induced apoptosis in cultured cells, p38 mitogen-activated protein kinase and p21/WAF1/Cip1, were examined. A marked increase in phosphorylation of p38 was observed within 1 h after Pc 4-PDT without changes in levels of the p38 protein. Levels of p21 were not altered in the xenografts in correspondence with the presence of mutant p53 in SW480 cells. Evaluation of tumor size showed that tumor growth resumed after a delay of 9-15 days. Our results suggest that: (a) Pc 4-PDT is effective in the treatment of SW480 human colon cancer xenografts independent of p53 status; (b) PARP cleavage may be mediated by caspase-9 and caspase-3 activation in the Pc 4-PDT-treated tumors; and (c) p38 phosphorylation may be a trigger of apoptosis in response to PDT in vivo in this tumor model.     

In vitro and in vivo inhibition of epidermal growth factor receptor-tyrosine kinase pathway by photodynamic therapy

PDT, a new therapeutic procedure for the management of many malignant conditions including skin cancer, involves the administration of a photosensitizing compound followed by illumination of the lesion with visible light. We earlier showed an involvement of: (i) WAF1/p21-cyclins (D1 and E)-cdk (2 and 6) network; and (ii) Rb/E2F-DP machinery during silicon phthalocyanine (Pc4)-PDT-mediated cell cycle dysregulation and apoptosis of human epidermoid carcinoma (A431) cells. Here, we investigated the involvement of EGFR-pathway during antiproliferative responses of Pc4-PDT in A431 cells and during ablation of murine skin papillomas. Pc4-PDT of A431 cells was found to result in a time-dependent down-modulation of the protein expression and phosphorylation of EGFR and Shc (an immediate downstream molecule in EGFR-pathway), during progressive increase in apoptotic response. To establish the relevance of these in vitro findings to in vivo situations, we subjected chemically- as well as ultraviolet B radiation-induced squamous papillomas in SENCAR and SKH-1 hairless mice, respectively, to Pc4-PDT, and assessed its effect on EGFR-pathway during ablation of these tumors. Pc4-PDT was found to result in a time-dependent: (i) inhibition of protein expressions of EGFR; and (ii) tyrosine phosphorylation of EGFR and Shc; and (iii) induction of apoptosis, during the regression of these tumors. These data suggest the involvement of EGFR-pathway during the antiproliferative effects of PDT. It is tempting to speculate that inhibitors of EGFR could enhance the therapeutic efficacy of PDT.     

Differential responses of Mcl-1 in photosensitized epithelial vs lymphoid-derived human cancer cells

The antiapoptotic Bcl-2-family proteins, Bcl-2 and Bcl-xL, are recognized phototargets of photodynamic therapy (PDT) with the mitochondrion-targeting phthalocyanine photosensitizer Pc 4. In the present study, we found that myeloid cell leukemia 1 (Mcl-1), another antiapoptotic member of the Bcl-2 family, was not photodamaged in Pc 4-PDT-treated human carcinoma cells MCF-7c3, MDA-MB468, DU145, and A431, although Mcl-1 turnover was observed after exposure of HeLa or MCF-7c3 cells to a supralethal dose of UVC. In contrast, when human lymphoma U937 and Jurkat cells were treated with Pc 4-PDT, staurosporine (STS) or UVC, Mcl-1 was cleaved to generate a 28-kDa fragment over a 2-4 h period. The cleavage of Mcl-1 was accompanied by the activation of caspases-3, -9, and -8. The broad-specificity caspase inhibitor z-VAD-fmk completely blocked Mcl-1 cleavage induced by PDT, STS or UVC, providing evidence for Mcl-1 as a substrate for caspases. Western blot analysis localized Mcl-1 to mitochondria, ER, and cytosol of both MCF-7c3 and U937 cells, suggesting that Mcl-1 protein, unlike Bcl-2 and Bcl-xL, is not a target for Pc 4-PDT, probably due to its localization to sites removed from those of Pc 4 binding. The 28-kDa cleaved fragment of Mcl-1, which has proapoptotic activity, was produced in PDT-treated lymphoid-derived cells, but not in cells of epithelial origin, suggesting that PDT-induced rapid and extensive apoptosis in lymphoma cells may result in part from the sensitivity of their Mcl-1 to caspase cleavage, removing an important negative control on apoptosis.     

Tailor-made approach to photodynamic therapy in the treatment of cancer based on Bcl-2 photodamage

It is very important to elucidate the mechanism of action and identify the molecular determinant of photodynamic medicine, in order to increase the number of clinical applications of photodynamic therapy (PDT) and perform personalized medicine. We have previously reported that PDT using some photosensitizers, such as phthalocyanine 4 (Pc 4) damages the anti-apoptotic protein Bcl-2, and that Bcl-2 is a molecular PDT target using a mitochondrion-targeting photosensitizer. In this study, we examined the molecular targets of Photofrin-PDT and NPe6-PDT, which are approved for early stage lung cancers by the Japanese Ministry of Health Labor and Welfare, by evaluating the photodamage to Bcl-2 using Western blot analysis. Our results showed that Photofrin-PDT damaged Bcl-2, induced morphologically typical apoptosis, and demonstrated equal sensitivity between MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) and Bcl-2 overexpressing cells, MCF-7c3-GFP-Bcl-2 cells, with a clonogenic assay. However, NPe6-PDT did not damage Bcl-2 and took longer to induce typical apoptosis compared with Photofrin-PDT. MCF-7c3-GFP-Bcl-2 cells were considerably more resistant to the lethal effects of NPe6-PDT than parental MCF-7c3 cells. In conclusion, Photofrin-PDT damages different molecular targets, and our data indicate that the extent of Bcl-2 photodamage can determine the sensitivity of cancer cells to apoptosis and to overall cell killing caused by PDT using Photofrin, but not the lysosomal targeting NPe6. The application of these findings to clinical PDT may depend on the levels of the Bcl-2 proteins in the tumor being treated, and the tailor-made medicine based on the Bcl-2 photodamage may overcome any resistance afforded by elevated amounts of Bcl-2.     

Photochemical destruction of the Bcl-2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4.

Photodynamic therapy (PDT), utilizing a photosensitizer and visible light, causes localized oxidative damage. With the mitochondrial photosensitizer Pc 4, PDT induces apoptosis, yet its molecular targets are not known. Here, the anti-apoptotic protein Bcl-2 is shown to be highly sensitive to PDT, as judged on Western blots by the disappearance of anti-Bcl-2-reactive material from the position of the native 26 kDa protein. The loss of Bcl-2 was PDT dose dependent and was observed for both endogenous and overexpressed Bcl-2 in several cell lines, immediately after PDT, and with chilled cells. It was accompanied by a trace of a 23-kDa cleavage product as well as high-molecular weight products that may result from photochemical crosslinking. PDT-induced Bcl-2 loss occurred in MCF-7 cells that do not express caspase-3 or in the presence of protease inhibitors, but was prevented, along with the induction of apoptosis, by the singlet oxygen scavenger L-histidine. Loss of FLAG-Bcl-2 was observed with both anti-FLAG and anti-Bcl-2 antibodies, indicating loss of native protein rather than simple BCL-2-epitope destruction. Photochemical damage was not observed in Bcl-x(L), Bax, Bad, the voltage-dependent anion channel, or the adenine nucleotide translocator. Therefore, Bcl-2 is one target of PDT with Pc 4, and PDT damage to Bcl-2 contributes to its efficient induction of apoptosis.     

Fluorescence resonance energy transfer reveals a binding site of a photosensitizer for photodynamic therapy

Phthalocyanine (Pc) 4, like many photosensitizers for photodynamic therapy (PDT), localizes to intracellular membranes, especially mitochondria. Pc 4-PDT photodamages Bcl-2 and Bcl-xL, antiapoptotic proteins interacting with the permeability transition pore complex that forms at contact sites between the inner and outer mitochondrial membranes. These complexes and the inner membrane are unique in containing the phospholipid cardiolipin. Nonyl-acridine orange (NAO) is a specific probe of cardiolipin. Here we show evidence for fluorescence resonance energy transfer from NAO to Pc 4, defining a binding site for the photosensitizer. This observation establishes an innovative tool for exploring the localization of other photosensitizers and additional fluorescent, mitochondrion-localizing drugs having appropriate spectral properties.     

Mechanism of cell death mediated by a BF2-chelated tetraaryl-azadipyrromethene photodynamic therapeutic: dissection of the apoptotic pathway in vitro and in vivo

Photodynamic therapy (PDT) is an established treatment modality for cancer. ADPM06 is an emerging non-porphyrin PDT agent which has been specifically designed for therapeutic application. Recently, we have demonstrated that ADPM06-PDT is well tolerated in vivo and elicits impressive complete response rates in various models of cancer when a short drug-light interval is applied. Herein, the mechanism of action of ADPM06-PDT in vitro and in vivo is outlined. Using a drug and light combination that reduces the clonogenicity of MDA-MB-231 cells by >90%, we detected a well-orchestrated apoptotic response accompanied by the activation of various caspases in vitro. The generation of reactive oxygen species (ROS) upon photosensitizer irradiation was found to be the key instigator in the observed apoptotic response, with the endoplasmic reticulum (ER) found to be the intracellular site of initial PDT damage, as determined by induction of a rapid ER stress response post-PDT. PDT-induced apoptosis was also found to be independent of p53 tumor suppressor status. A robust therapeutic response in vivo was demonstrated, with a substantial reduction in tumor proliferation observed, as well as a rapid induction of apoptosis and initiation of ER stress, mirroring numerous aspects of the mechanism of action of ADPM06 in vitro. Finally, using a combination of (18) F-labeled 3'-deoxy-3'-fluorothymidine ((18) F-FLT) nuclear and optical imaging, a considerable decrease in tumor proliferation over 24-hr in two models of human cancer was observed. Taken together, this data clearly establishes ADPM06 as an exciting novel PDT agent with significant potential for further translational development.     

Bax is essential for mitochondrion-mediated apoptosis but not for cell death caused by photodynamic therapy

The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF-7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.     

Phthalocyanine 4 photodynamic therapy-induced apoptosis of mouse L5178Y-R cells results from a delayed but extensive release of cytochrome c from mitochondria

Photodynamic therapy (PDT) activates the mitochondrial pathway of apoptosis, for which the release of cytochrome c into the cytosol is considered critical. To further elucidate the role of cytochrome c release in PDT-induced apoptosis, we monitored cytochrome c localization immunocytochemically and related it to nuclear apoptosis of the same cells. When mouse L5178Y-R cells were treated with 300 nM phthalocyanine (Pc) 4 and 0-75 mJ/cm(2) red light, cytochrome c release had a dose response similar to that of clonogenic cell killing, with nearly identical threshold doses. Within individual cells, the release of cytochrome c appeared to be an all-or-none phenomenon. Moreover, it was tightly associated with activation of a caspase-3-like protease and changes in nuclear morphology. Thus, in response to Pc 4-PDT, the release of cytochrome c from mitochondria is a key determinant of apoptotic cell death.     

Up-regulation of clusterin during phthalocyanine 4 photodynamic therapy-mediated apoptosis of tumor cells and ablation of mouse skin tumors

Photodynamic therapy (PDT) using the silicon phthalocyanine photo-sensitizer Pc 4 is an oxidative stress associated with the induction of apoptosis in many cancer cells in vitro and in vivo. The mechanisms of PDT-induced tumor cell killing leading to apoptosis are incompletely understood. Clusterin, a widely expressed glycoprotein, is induced in tissues regressing as a consequence of oxidative stress-mediated cell death. Treatment of apoptosis-sensitive human epidermoid carcinoma cells (A431) with PDT resulted in significant up-regulation of clusterin with a maximum at 12 h after treatment, whereas clusterin levels in Pc 4-PDT-treated, apoptosis-resistant, radiation-induced fibrosarcoma (RIF-1) cells remained unchanged. The i.v. administration of Pc 4 to mice bearing chemically or UVB radiation-induced skin papillomas, followed by light application, led to increased clusterin protein expression, peaking 24 h after the treatment, when tumor regression was apparently visible. These data, for the first time, demonstrate the involvement of clusterin in PDT-mediated cell death and during tumor regression. This may have relevance in improving the efficacy of PDT using pharmacological inducers of clusterin.     

Lysosomal cathepsin initiates apoptosis, which is regulated by photodamage to Bcl-2 at mitochondria in photodynamic therapy using a novel photosensitizer, ATX-s10 (Na)

ATX-s10 is a novel and second-generation photosensitizer for photodynamic therapy (PDT). In order to conduct clinical trials of ATX-s10-PDT and/or extend its clinical applications, it is very important to elucidate the mechanisms of the action of ATX-s10-PDT. We examined the apoptic response against ATX-s10-PDT using a Bcl-2 or Bcl-2 mutant overexpressing cells. Using fluorescent microscopy, ATX-s10 localized not only to mitochondria but also to lysosomes and possibly other intracellular organelles, but not to the plasma membrane or the nucleus. These results suggest that ATX-s10-PDT can damage mitochondria and lysosomes. By Western blot analysis, ATX-s10-PDT damaged Bcl-2, which localized preferentially at mitochondrial membranes, and caused Bcl-2 to cross-link immediately after laser irradiation. However, ATX-s10-PDT was not able to rapidly induce morphologically typical apoptosis (i.e. chromatin condensation and fragmentation) as PDT using mitochondria targeted photosensitizers, such as phthalocyanine 4 (Pc 4). Pharmacological inhibitions of lysosomal cytokine protease cathepsins, such as cathepsin B and D, protected MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) from apoptosis caused by ATX-s10-PDT. Overexpression of wild-type Bcl-2 or Bcl-2Delta33-54 resulted in relative resistance of cells to ATX-s10-PDT, as assessed by the degree of morphological apoptosis or loss of clonogenicity. We conclude that lysosomal damage by ATX-s10-PDT can initiate apoptotic response and this apoptotic pathway can be regulated by photodamage to Bcl-2 via mitochondrial damage.     

Dissociation of mitochondrial depolarization from cytochrome c release during apoptosis induced by photodynamic therapy

Photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 induces rapid apoptosis in mouse lymphoma (LY-R) cells, initiating with the release of cytochrome c from mitochondria. It has been proposed that the opening of the mitochondrial membrane permeability transition pores, which results in the dissipation of the mitochondrial membrane potential (Deltapsi(m)), is essential for the escape of cytochrome c from mitochondria into the cytosol as well as for apoptotic cell death. Therefore, we have assessed the correlation between the loss of Deltapsi(m)and the release of cytochrome c following PDT. Treatment of LY-R cells with 300 nM Pc 4 and 60, 90 or 120 mJ/cm(2)of red light resulted in apoptosis of 80-90% of the cells, accompanied by >20-fold elevation in caspase-3-like activity within one h. At all 3 doses of PDT employed here, the majority of the cytochrome c was released from mitochondria at 15 min after irradiation, as determined by an immunohistochemical method. In contrast, the loss of Deltapsi(m)following PDT, as monitored by the uptake of JC-1 or Rh-123, depended on the PDT dose and the post-treatment time. In spite of the release of cytochrome c at 15 min after each of the 3 doses, a corresponding loss of Deltapsi(m)was observed only for those cells that received the highest dose of PDT. Virtually all cells that received one of the lower doses of PDT (300 nM Pc 4 plus 60 or 90 mJ/cm(2)) maintained normal Deltapsi(m). Hence, our results support the conclusion that the release of cytochrome c from mitochondria resulting from Pc 4-PDT-induced photodamage is independent of the loss of Deltapsi(m). Therefore, it is important to consider a range of doses of this or other apoptotic stimuli in deciphering the relationship of metabolic responses that contribute to apoptosis.     

High expression of GADD-45alpha and VEGF induced tumor recurrence via upregulation of IL-2 after photodynamic therapy using NPe6

NPe6 is a novel second-generation photosensitizer used for photodynamic therapy (PDT). PDT using NPe6 and diode laser (664 nm) induces cell death, inflammatory reactions, immunological responses and damage to the microvasculature. In this study, we evaluated the influence of the immunological responses and of enhanced angiogenesis on the anti-tumor effect of NPe6-PDT using cytokine-overexpressing Lewis lung carcinoma (LLC), LLC-IL-2 cells both in vitro and in vivo. We showed by DNA microarray analysis in vitro that IL-2 and GADD-45alpha (growth arrest and DNA damage 45 alpha) mRNA expressions were induced by 3 h after NPe6-PDT applied at a dose killing 90% of the cells (LD90). IL-2-overexpressing cells (LLC/IL-2 cells) were resistant to the loss of clonogenicity as compared to the parental LLC cells in vitro. Furthermore, in female C57BL/6 mice, NPe6-PDT produced a cure rate of 66.7% in LLC tumors, whereas the cure rate was only 16.6% in LLC/IL-2 tumors, and overexpression of IL-2 caused failure of NPe6-PDT, with tumor recurrence, in vivo. These results suggest that IL-2 expression may play an unfavorable role in attenuation of the antitumor effect of NPe6-PDT. It has been reported that the expression of vascular endothelial growth factor (VEGF), in particular, may cause tumor recurrence after PDT and exert unfavorable effect in relation to attenuate the anti-tumor activity of PDT. Results of immunohistochemical analysis of LLC/IL-2 tumors have revealed that the expressions of GADD-45alpha and VEGF are induced in these tumors after PDT, and in particular, 12 h after PDT, the expression levels were much higher as compared with those in the LLC tumors. The results of our studies using in vitro and in vivo models suggest that the cell death caused by PDT was inhibited by induction of GADD-45alpha expression and that tumor recurrence was promoted by the enhancement of VEGF expression mediated by IL-2 upregulation. Therefore, it is speculated that the use of an IL-2 inhibitor may improve the efficacy of NPe6-PDT.     

Involvement of Fas receptor and not tumor necrosis factor-alpha receptor in ultraviolet-induced activation of acid sphingomyelinase

Fas receptor and tumor necrosis factor receptor-1 (TNFR1) mediate the activation of acid sphingomyelinase (ASMase), which catalyzes the hydrolysis of sphingomyelin to ceramide. Ceramide acts as a second messenger in mediating cell growth, differentiation, stress response, and apoptosis. Ultraviolet (UV) irradiation induces Fas receptor and TNFR1 aggregation. However, the roles of Fas receptor and TNFR1 in mediating UV-induced ASMase activation have not been explored. In this report, we demonstrate that Fas receptor, not TNFR1, mediated UV-induced activation of ASMase. Our data indicate that ASMase activity was not induced with UV irradiation but by TNFalpha in MCF-7 cells that expressed low levels of Fas receptor. In contrast, ASMase was activated by UV irradiation or TNFalpha treatment in Fas stably transfected MCF-7 cells. Immunofluorescence staining of TNFR1 on MCF-7 cells showed that TNFR1 was aggregated after treatment with UV irradiation or TNFalpha. However, UV-induced aggregation of TNFR1 did not lead to induction of ASMase activity. These results suggest that Fas receptor aggregation is solely responsible for UV-induced activation of ASMase. Further, with the use of BJAB and dominant-negative Fas-associated death domain-containing protein (FADD) stably transfected BJAB cells, we demonstrated that dominant-negative FADD partly inhibited UV-induced ASMase activation. Our results suggest that FADD is involved in UV-induced and Fas-mediated signaling pathways for activation of ASMase. Mol. Carcinog. 30:47-55, 2001.     

Photodynamic therapy of human breast cancer xenografts lacking caspase-3

The human breast cancer cell line MCF-7 is deficient in procaspase-3 and in caspase-3-dependent steps in apoptosis due to deletion of the CASP-3 gene. We previously found that the cells transfected with empty vector (MCF-7v cells) were considerably less sensitive to photodynamic treatment in vitro with the phthalocyanine photosensitizer Pc 4 than were the cells stably transfected with human procaspase-3 cDNA (MCF-7c3 cells); however, overall cell killing, as determined by a clonogenic assay, was not affected by the presence of procaspase-3. The present study was undertaken to determine whether photodynamic therapy (PDT) in vivo was dependent on the ability of the cells to carry out the late steps in apoptosis that are catalyzed by this caspase. Xenografts of MCF-7 cells and the isogenic-derived MCF-7v and MCF-7c3 cells were generated in female athymic nude mice implanted with an estrogen pellet. MCF-7c3 xenografts, but not those of the other two lines, continued to express procaspase-3, as revealed by Western blots of proteins from the cells and the xenografts. When the xenografts reached 50-120 mm(3), some were treated with PDT (1mg/kg Pc 4 i.v. followed 48 h later by 150 J/cm(2) light at 672 nm and 150 mW/cm(2)), while others served as controls (no treatment, light alone, or Pc 4 alone). All Pc 4-PDT-treated tumors and none of the controls exhibited either complete or strong partial responses, and complete responses were durable for the entire observation period of 16 days. The responses were not dependent upon the presence of procaspase-3 in the xenografts. The results indicate that the rapid response of Pc 4-PDT-treated tumors in vivo is not due to their ability to carry out the major caspase-3-mediated late steps in apoptosis.     

Tissue distribution and photosensitizing properties of mono-L-aspartyl chlorin e6 in a mouse tumor model

Mono-L-aspartyl chlorin e6 (NPe6) is a photosensitizer that possesses properties such as chemical purity and a major absorption band at 664 nm which are potentially exploitable for photodynamic therapy (PDT). The current investigation examined pharmacological and photosensitizing parameters of NPe6 in tumor and normal tissues in mice. [14C]NPe6 was used to obtain quantitative tissue distributions of the photosensitizer as a function of: (a) time following administration; (b) drug dose; (c) mode of drug administration; and (d) tumor size. The in vivo photosensitizing efficiency of NPe6 was compared directly to Photofrin II in experiments which evaluated tumor responses and induction of normal skin damage. Initial PDT experiments demonstrated that NPe6 was ineffective at inducing tumor cures when a 24-h time interval (between drug administration and light treatment) was used. However, PDT-induced tumor cures were obtained when NPe6 was administered 4-6 h prior to light exposure, and these NPe6-PDT treatment parameters were as effective as standard Photofrin II-mediated PDT. Interestingly, the level of PDT-induced normal skin damage was significantly greater for Photofrin II than for NPe6 at comparable drug and light doses. An analysis of pharmacological data and PDT time interval requirements suggests that plasma concentrations of NPe6 may be a more important predictive factor than tumor tissue levels of the photosensitizer for the production of PDT-mediated tumor cures. The results of this investigation indicate that NPe6 is an effective tumor photosensitizer with in vivo clearance properties that eliminate the side effect of prolonged normal skin photosensitization.     

A possible relationship between the anti-cancer potency of photodynamic therapy using the novel photosensitizer ATX-s10-Na(II) and expression of the vascular endothelial growth factor in vivo

ATX-s10-Na(II) is a novel second-generation photo-sensitizer for photodynamic therapy (PDT). PDT using ATX-s10 and diode laser (670 nm) induces an apoptotic response, inflammatory reaction, immune reaction and damage to the microvasculature. In particular, the vascular shut-down effect plays an important role in the anti-tumor activity of ATX-s10-PDT. It has been reported that PDT induces hypoxia and expression of the vascular endothelial growth factor (VEGF) via the hypoxia-inducible factor 1 (HIF1)-alpha pathway. We hypothesized that the expression of VEGF may cause tumor recurrence after PDT and exert unfavorable effect against the anti-tumor activity of ATX-s10-PDT. In this study, we showed by DNA microarray analysis in vitro that VEGF mRNA expression was induced 3 h after laser irradiation in ATX-s10-PDT. We compared the anti-tumor activity of ATX-s10-PDT against lung cancer cell lines SBC-3 and SBC-3/VEGF, the latter overexpressing VEGF; there was no significant difference in the sensitivity to the PDT between the two cell lines as assessed by clonogenic assay. Furthermore, no statistically significant difference in the anti-tumor effect of PDT, as measured by tumor cures, was found between SBC-3 and SBC-3/VEGF tumors in female Balb/c-nu/nu nude mice in vivo. In conclusion, ATX-s10-PDT may prevent tumor recurrence despite induction of VEGF and promotion of tumor angiogenesis, which are known to enhance tumor proliferation and survival.