Involvement of nitric oxide during phthalocyanine (Pc4) photodynamic therapy-mediated apoptosis

Photodynamic therapy (PDT), a new treatment modality, uses a combination of photosensitizing agent and visible light for the therapy of many solid malignancies. The hallmark of PDT is intracellular oxidative stress mediated by reactive oxygen species, which, through a cascade of events, results in a cell kill that induces apoptosis in some cells. To better understand the mechanism of apoptosis, we hypothesized the role of nitric oxide (NO), which is considered to be involved in a variety of physiological and pathological processes, during PDT. The model photosensitizer we have been working with is a silicon-phthalocyanine compound termed Pc4. Here, we investigated the involvement of NO during Pc4 PDT in PDT of apoptosis-resistant radiation-induced fibrosarcoma (RIF-1) cells and in PDT of apoptosis-sensitive human epidermoid carcinoma (A431) cells. Pc4 PDT resulted in a rapid increase in nitrite production in A431 cells, starting as early as 15 s post-PDT, and showed a progressive increase up to 15 min post-PDT. This increase in nitrite production was observed in cell lysates as well as in the cell culture medium. RIF-1 cells did not show an increase in nitrite production in either the cell lysates or the culture medium. At this time, a majority of the cells were viable. The Western blot analysis also showed a rapid increase in the expression of the constitutive form of NO synthase as early as 15 s post-PDT when compared to that of the controls. This response showed a dose dependency up to 5 min after Pc4 PDT. This observation was confirmed by a [3H]L-citrulline assay, which also showed a similar pattern for constitutive NO-synthase activity. RIF-1 cells did not show any change in protein expression or enzyme activity after the same treatment. These data, for the first time, demonstrate the generation of NO during PDT and suggest that it may be involved in PDT-mediated apoptosis. This may have relevance in improving the therapeutic efficacy of PDT using pharmacological modulators of NO or NO synthase.