Signaling events in apoptotic photokilling of 5-aminolevulinic acid-treated tumor cells: Inhibitory effects of nitric oxide

Abstract

Antitumor photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that can destroy tumor and tumor vasculature cells. NO produced by these cells could be pro-carcinogenic by inhibiting apoptosis and promoting angiogenesis and tumor growth. We recently showed that NO from a chemical donor or activated macrophages makes COH-BR1 breast tumor cells more resistant to photokilling sensitized by 5-aminolevulinic acid (ALA)-generated protoporphyrin IX (PpIX). Signaling events associated with this hyperresistance have now been examined. ALA-treated COH-BR1 cells containing mitochondria-localized PpIX died mainly by apoptosis after being irradiated. Underlying redox signaling associated with MAP kinase (ERK1/2, p38, JUN) phosphorylation-activation and heme oxygenase-1 (HO-1) upregulation was studied using immunoprecipitation and Western blot methodology. ALA/light treatment resulted in activation of pro-apoptotic JNK and p38α, and deactivation of pro-survival p38β and ERK1/2. Involvement of both JNK and p38 in apoptosis was established by using a specific inhibitor for each. Spermine NONOate-derived NO, introduced immediately before irradiation, provided substantial protection against apoptosis. This was accompanied by greater HO-1 induction and strong inhibition of each MAP kinase effect seen in the absence of NO. Downstream of JNK and p38α activation, a marked upregulation/activation of proapoptotic Bax and Bid was observed along with down-regulation of anti-apoptotic Bcl-xL, each response being reversed by NO. These findings provide new insights into signaling activity associated with the intrinsic apoptotic pathway in ALA-PDT and how this activity can be modulated by NO.

Accelerated migration and invasion of prostate cancer cells after a photodynamic therapy-like challenge: Role of nitric oxide

“…When an iNOS inhibitor (1400W) or NO scavenger (cPTIO) was used, it was introduced 30 min before ALA and maintained at the same concentration throughout irradiation and post-irradiation incubation. Immediately after ALA treatment, cells were switched to fresh medium either lacking or containing iNOS inhibitor or NO scavenger, and then irradiated on a translucent plastic platform over a bank of four 40-W cool-white fluorescent tubes [22]. The intensity (fluence rate) of light reaching the bottom surface of culture dishes was ~11 W/m 2 , as measured with a YSI radiometer (Yellow Springs, OH).…”

“…2.3 and then either not irradiated (dark control) or irradiated (~1 J/cm 2 ). Cell lysates were prepared as described [21,22], analyzed for total protein, then subjected to Laemmli SDS-PAGE, using 15% acrylamide/bis-acrylamide. Separated proteins were transferred to a polyvinyidene difluoride membrane and, after blocking, the membrane was incubated overnight at 4 °C with an iNOS, TIMP1, TIMP-2, integrin α6, or integrin β1 antibody at a supplier-recommended dilution.…”

The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

“…Di Venosa et al 19 , however, did not observe any relationships between NO production and photodynamic cell injury. The cytoprotection, anti-apoptotic effect of NOS in 5-aminolevulinic-mediated PDT of both murine tumor and normal cells, has been reported 20 , and iNOS was shown to play the leading role in it 21 . Another view was suggested by Lu et al 22 : the authors suppose that mitochondrial NOS and following cytochrome c release play the leading role is PDT induced cell damage, in particular, in the case of cancer cells.…”

Breast cancer as photodynamic therapy target: Enhanced therapeutic efficiency by overview of tumor complexity

“…NO produced by photosensitized cells could be pro-carcinogenic by inhibiting apoptosis. It was shown that NO from chemical donor or activated macrophages made breast tumor cells sensitized by 5-aminolevulinic acid-generated protoporphyrin IX more resistant to photo killing by providing substantial protection against apoptosis [39,40] . Additionally, it was demonstrated that PDT-treated breast cancer cells acquired the ability to upregulate inducible-nitric oxide synthase (iNOS) expression [41] .…”