Photodynamic therapy.

Cytokines induce the mouse macrophage cell line RAW 264.7 to express cytokine-inducible nitric oxide synthase (iNOS), which is active only in dimeric form. Because dimerization of purified iNOS subunits requires tetrahydrobiopterin, heme, and L-arginine, we investigated if availability of these factors also influences intracellular assembly of dimeric iNOS. Following exposure to cytokines, iNOS protein was found to accumulate in a near linear manner over 16 h of further culture. In contrast, dimeric iNOS accumulated at a slower rate that continuously decreased during culture, resulting in only 25% of the accumulated iNOS protein being in dimeric form by 16 h. Further experiments argued against dimer instability or L-arginine and tetrahydrobiopterin availability as factors limiting iNOS dimer accumulation. Blocking cellular NO synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) greatly increased iNOS dimer assembly, indicating NO synthesis limited iNOS dimerization. NO synthesis was found to prevent an increase in soluble heme level that was associated with iNOS induction in Nomega-nitro-L-arginine methyl ester-treated cells and also diminished heme insertion into iNOS. These NO-related defects were not reversed by adding heme precursors or hemin to the activated cell cultures. Measurement of iron release from activated cells demonstrated that endogenous NO synthesis substantially increased the release of 59Fe to the medium. These observations suggest that iNOS dimerization is limited to a large extent by iNOS NO synthesis. NO appears to limit intracellular assembly of dimeric iNOS by preventing heme insertion and decreasing heme availability.

Aminolevulinic Acid↗

PubMed1996

Induction of tumor necrosis by delta-aminolevulinic acid and 1,10-phenanthroline photodynamic therapy.

delta-Aminolevulinic acid (ALA) causes cells to accumulate protoporphyrin IX (Proto) and heme. Exposure to light in vitro causes intracellular Proto to initiate formation of singlet oxygen molecules, leading to self-destruction. This photoactivated destruction by ALA in vitro is enhanced by addition of the tetrapyrrole modulator 1,10-phenanthroline (Oph), which increases cellular accumulation of Proto. Here we significantly extend this idea by evaluating the efficacy of ALA and Oph photodynamic therapy of solid tumors in vivo. Methylcholanthrene-induced fibrosarcoma (Meth-A) cells were used, which lead to the formation of solid tumors when implanted into syngeneic recipients. Initially, suspensions of Meth-A cells were treated in vitro with combinations of ALA and Oph. Meth-A cells in suspension accumulated 6-fold greater amounts of Proto (P < 0.05) after 3-h incubation with ALA and Oph than when incubated with ALA alone, and were also more susceptible to subsequent photoactivated cell lysis in vitro. Similarly, solid Meth-A tumors grown in syngeneic BALB/c mice accumulated significant (P < 0.05) amounts of Proto 3 h after in vivo treatment with ALA, and Oph synergized with ALA to significantly (P < 0.05) enhance the induction of Proto in these tumors. ALA and Oph-based phototreatment of mice bearing Meth-A solid tumors resulted in necrosis of tumors, as determined by a significant reduction in both size and histopathology, with little damage to surrounding normal tissue. These data directly demonstrate the experimental usefulness of Proto modulators for ALA-based photodynamic therapy in the treatment of solid tumors in vivo and provide a rationale for their potential application in a multitude of tumor types.

Aminolevulinic Acid↗