Apoptosis Mechanisms Related to the Increased Sensitivity of Jurkat T‐cells vs A431 Epidermoid Cells to Photodynamic Therapy with the Phthalocyanine Pc 4^†‡

Abstract

To examine the clinical applicability of Pc 4, a promising second-generation photosensitizer, for the photodynamic treatment of lymphocyte-mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte-derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose-response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4-PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl-2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl-2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl-1 was extensively cleaved in a dose- and caspase-dependent manner in Jurkat, but not in A431, cells exposed to Pc 4-PDT. Thus, the greater killing by Pc 4-PDT in Jurkat compared with A431 cells correlated with greater Bcl-2 photodamage and more strongly to the more extensive Mcl-1 degradation. Pc 4-PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T-cell-mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl-2 family proteins, especially Mcl-1, in the therapeutic response.

Activated T cells exhibit increased uptake of silicon phthalocyanine Pc 4 and increased susceptibility to Pc 4-photodynamic therapy-mediated cell death

“…Cell types and states of activation vary significantly in their susceptibility to oxidative stress, which may account for our previous observation that a human acute T-cell leukemia cell line (Jurkat) was far more susceptible to Pc 4-PDT-induced cell death than was a human epidermoid carcinoma cell line (A431) (15). Interestingly, within populations of normal human T cells, we observed a hierarchy in which the highest uptake of Pc 4 occurred in activated T cells with large numbers of mitochondria, the next highest uptake was in resting T cells, with the lowest Pc 4 uptake occurring in Treg.…”

“…The increased numbers of mitochondria associated with activated T cells suggests that a mitochondrion-induced apoptosis may occur in T cells as observed for other cell types. Specifically, Pc 4-PDT has been demonstrated to damage the anti-apoptotic proteins Bcl-2 and Bcl-XL when they are bound to the mitochondrial or ER membranes, resulting in Bax translocation from the cytosol to mitochondria, and the release of cytochrome c and induction of the intrinsic apoptosis pathway that may be enhanced by lysosomal disruption (46, 15, 47–51). …”

“…Pc 4 has been shown to preferentially bind to mitochondrial, lysosomal and endoplasmic reticulum (ER) membranes in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro (15, 16) as well as in numerous other cancer cell lines (reviewed in ref. 15).…”

“…15). However, whether or not there is differential uptake and function as PDT on primary human, resting, activated, and malignant T cells is unknown.…”

Monitoring Pc 4 photodynamic therapy in clinical trials of cutaneous T-cell lymphoma using noninvasive spectroscopy

“…In preclinical studies, T-cells were found to be more susceptible than keratinocytes to Pc 4-PDT-induced-apoptosis. 4 Accordingly, patients with early stage MF malignancies were enrolled in a phase I clinical trial of topical Pc 4-PDT at CWRU. Results of the study are not yet published, but preliminary observations are encouraging with respect to safety and possible biological response.…”