Abstract Purpose: Most studies accept a multistep pathogenic process in melanoma that may include the phases of benign nevi and dysplastic nevi, melanoma, and metastatic melanoma. Dysregulation of cellular proliferation and apoptosis is probably involved in melanoma progression and response to therapy. We have studied the expression of galectin-3, a β-galactoside-binding protein involved in apoptosis, angiogenesis, and cell proliferation, in a large series of melanocytic lesions, and correlated the expression with clinical and histologic features. Experimental Design: Tissue microarray blocks of 94 melanocytic lesions were semiquantitatively evaluated by immunohistochemistry for the cytoplasmic or nuclear expression of galectin-3. Results: Primary and metastatic melanomas expressed galectin-3 at a significantly higher level than nevi in both cytoplasm and nuclei (P < 0.0073). There was a significant association between anatomic source (as indirect indication of level of sun-exposure) and cytoplasmic and nuclear expression. Lymph node and visceral metastases had a higher level of expression than s.c. lesions (P < 0.004). Interestingly, there was an almost significant finding of worse survival in those patients with lesions showing higher levels of cytoplasmic than nuclear galectin-3 expression (log-rank test, P = 0.06). Conclusions: Melanocytes accumulate galectin-3 with tumor progression, particularly in the nucleus. The strong association of cytoplasmic and nuclear expression in lesions of sun-exposed areas suggests an involvement of UV light in activation of galectin-3.
ABSTRACT The photobleaching of meta ‐tetra(hydroxyphenyl)chlorin m THPC) (irradiation wavelength 413 nm) in protein‐containing solution was evaluated by decay in absorbance in Soret band and in fluorescence (λ exc = 423 nm, λ em = 655 nm). Light exposure resulted in a decrease in absorption throughout the spectrum and simultaneous appearance of new absorption bands in the spectral region 325–450 nm. The rate of m THPC photodegradation, followed by decay in absorbance, was 15‐fold lower than that observed in fluorescence. This fact reflects the photobleaching of presumably monomeric, fluorescing species of m THPC. In order to determine the consequences of photobleaching of fluorescing m THPC material on cellular uptake and photocytotoxicity, human HT29 colon adenocarcinoma cells were incubated with photobleached m THPC during 5 h with or without following irradiation with the fixed fluence. Surprisingly, but up to the time when the fluorescence decreased by 50%, only a slight decrease in photocytotoxicity was detected. Either aggregated forms that have been taken up undergo intracellular monomerization (but we did not observe increase in fluorescence in living cells) or the photodynamic activity is mostly due to aggregates. The discrepancy of m ‐THPC‐photodynamic therapy (PDT) effect and fluorescence measurements may suggest that aggregated m ‐THPC plays an important role in m THPC‐PDT.
La therapie photodynamique (PDT) est une modalite d'interet croissant de traitement des tumeurs accessibles a la lumiere. Elle est basee sur l'administration d'un photosensibilisant qui se localise dans les tumeurs et genere des radicaux libres toxiques (reaction photochimique de type 1) ou de l'oxygene singulet (type II) suite a une irradiation lumineuse. Des etudes du mecanisme d'action et de photodegradation d'un photosensibilisant de deuxieme generation, la meta-tetrahydroxyphenyIchlorine (mTHPC) ont ete conduites afin d'ameliorer son efficacite. Le suivi spectroscopique de la photodegradation de la mTHPC a revele un photoblanchiment preferentiel de ses formes fluorescentes. L'elimination des fractions fluorescentes par preirradiation n'a induit qu'une perte negligeable de l'activite phototoxique, suggerant que les fractions agregees de la mTHPC contribuent aux dommages cellulaires. L'examen in vitro des mecanismes photodynamiques de destruction cellulaire avec la mTHPC a revele que ce processus depend de la presence d'oxygene moleculaire. L'utilisation de piegeurs des especes reactives de l'oxygene a revele que la photosensibilisation par la mTHPC est essentiellement due a la production d'oxygene singulet. Les reactions de photoperoxydation jouent un role mineur dans la photocytotoxicite induite par la mTHPC. Les cellules d'adenocarcinome de colon humain HT29 incorporent efficacement l'antioxydant a-tocopherol in vitro, contrairement aux fibroblastes normaux humains MRC-5. L'a-tocopherol et son analogue hydrosoluble, le Trolox, augmentent les dommages induits par la mTHPC sur les cellules tumorales, mais pas sur les cellules normales. L'administration de Trolox in vivo (250 mg/kg) 1 heure 30 avant l'irradiation augmente l'effet de la PDT avec la mTHPC sur les xenogreffes de tumeurs humaines. Cet effet necessite la presence de Trolox durant la phase photochimique. Des experiences de photolyse par eclair laser ont montre que le Trolox reagit avec la mTHPC a l'etat triplet, en formant un anion radical de la mTHPC. Des analyses cinetiques ont suggere que la voie radicalaire favorisee par le Trolox peut agir en relais avec la voie de l'oxygene singulet en cas de conditions hypoxiques. Ces etudes montrent que la PDT est sujette a une modulation photochimique par les antioxydants.
The photobleaching of meta-tetra(hydroxyphenyl)chlorin mTHPC) (irradiation wavelength 413 nm) in protein-containing solution was evaluated by decay in absorbance in Soret band and in fluorescence (λexc= 423 nm, λem= 655 nm). Light exposure resulted in a decrease in absorption throughout the spectrum and simultaneous appearance of new absorption bands in the spectral region 325–450 nm. The rate of mTHPC photodegradation, followed by decay in absorbance, was 15-fold lower than that observed in fluorescence. This fact reflects the photobleaching of presumably monomeric, fluorescing species of mTHPC. In order to determine the consequences of photobleaching of fluorescing mTHPC material on cellular uptake and photocytotoxicity, human HT29 colon adenocarcinoma cells were incubated with photobleached mTHPC during 5 h with or without following irradiation with the fixed fluence. Surprisingly, but up to the time when the fluorescence decreased by 50%, only a slight decrease in photocytotoxicity was detected. Either aggregated forms that have been taken up undergo intracellular monomerization (but we did not observe increase in fluorescence in living cells) or the photodynamic activity is mostly due to aggregates. The discrepancy of m-THPC-photodynamic therapy (PDT) effect and fluorescence measurements may suggest that aggregated m-THPC plays an important role in mTHPC-PDT.
Photodynamic activation of macrophage-like cells contributes to an effective outcome of photodynamic therapy (PDT) treatment. The possibility for an enhancement of macrophage activity by photosensitization with meta-tetra(hydroxyphenyl)chlorin (mTHPC) (1 μg ml–1) was studied in U937, monocyte cell line differentiated into macrophages (U937Φ cells). Phagocytic activity of U937Φ cells was evaluated by flow-cytometry monitoring of ingestion of fluorescein-labelled Escherichia coli particles. Increase in irradiation fluence up to 3.45 mJ cm–2 (corresponding irradiation time 15 s) resulted in significant increase in fluorescence signal (145%), while at higher light fluences the signal lowered down to the untreated control values. A light energy-dependent production of tumour necrosis factor-alpha (TNF-α) by photosensitized macrophages was further demonstrated using the L929 assay. The maximum TNF-α mediated cytolysis was observed at 28 mJ cm–2 and was 1.7-fold greater than that in control. In addition, we demonstrated a fluence-dependent increase in nitric oxide (NO) production by mTHPC-photosensitized macrophages. NO release increased gradually and reached a plateau after irradiation fluence of 6.9 mJ cm–2. Cytotoxicity measurements indicated that the observed manifestations of mTHPC-photosensitized macrophage activation took place under the sublethal light doses. The relevance of the present findings to clinical mTHPC-PDT is discussed.
