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ANTAGONISTIC EFFECTS OF ENDOGENOUS NO IN GLIOBLASTOMA PDT MODELS
[46]
[45]
About 20 years ago, Henderson et al. and Korbelik et al. , using various mouse syngeneic tumor models
(e.g., RIF, SCCVII, and EMT6) and Photofrin® as PS, were the first to determine how endogenous NO
might affect PDT efficacy in vivo. They showed that PDT cure rate could be significantly improved when
NG-nitro-L-arginine (L-NAME), a non-specific inhibitor of NOS activity, was administered immediately
after irradiation. A striking correlation was made between NO output and extent of improvement with
L-NAME: tumors with the highest output responded best and those with the lowest output worst . It was
[46]
concluded that endogenous NO signaled for increased tumor resistance to PDT repression and did so in a
NO dose-dependent manner. The L-NAME effects were principally attributed to NO’s vasodilatory effects
acting in opposition to PDT’s known constrictive effects on the tumor microvasculature [45,46] . Follow-up
[47]
studies by Reeves et al. , using ALA-induced PpIX as PDT sensitizer for mouse RIF and EMT6 tumors,
confirmed the above findings and again concluded that endogenous NO, by opposing vascular damage,
can significantly increase tumor resistance to PDT. Although these studies [45-47 ] and more recent ones by
[48]
Rapozzi et al. clearly established that NO can antagonize PDT, several key questions were left largely
unsettled, which include: (1) whether this NO is generated by tumor cells per se, proximal endothelial
cells, macrophages, fibroblasts, or possibly all of these; (2) which NOS isoform plays a dominant role in any
given tumor; (3) whether the NOS/NO in question acts at a pre-existing level or is upregulated in response
to PDT stress; and (4) the signaling mechanisms involved in NOS expression and NO-induced resistance.
Over the past ten years, the authors and lab colleagues have focused on these questions using various
cancer cell lines, including glioblastoma lines. Key findings from this work are discussed below.

Hyper-resistance imposed by photostress-upregulated iNOS/NO
As indicated above, PDT can often circumvent any innate or acquired tumor resistance to conventional
chemotherapy or radiotherapy. It is now clear, however, that resistance mechanisms also exist for PDT,
some of which are acquired during treatment. For example, there is evidence that activity of cytosolic
ROS scavenging enzymes such as type-1 glutathione peroxidase and catalase are increased in lymphocytes
subjected to a modest photodynamic challenge . In addition, many cancer cell types, including
[49]
glioblastomas, can export PpIX and other PS via the ABCG2 transporter, inhibition of which increases
photosensitivity [44,50] . Another PDT resistance mechanism, which was discovered in the authors’ laboratory,
involves NO generated specifically by tumor cell iNOS, particularly that which is upregulated in response
to PDT stress [51-55] . This was demonstrated in recent experiments carried out on human glioblastoma
[56]
U87-MG and U251-MG cells (henceforth referred to as U87 and U251) . As shown in Figure 2A, U87
cells sensitized in mitochondria with ALA-induced PpIX were progressively inactivated after exposure
2
to increasing fluences of broad-band visible light, 4 J/cm , reducing the viable fraction by ~45% 20 h
[56]
after irradiation . ALA alone or light alone was completely innocuous. When added before ALA/light
treatment, 1400W (an enzyme inhibitor with a high specificity for iNOS) increased the extent of cell
photokilling throughout, as did the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-
1-oxyl-3-oxide (cPTIO) [Figure 2A]. Similar results were obtained with U251 cells [Figure 2B]. ALA/
light-induced U87 or U251 cell death occurred primarily via intrinsic (mitochondria-initiated) apoptosis,
as assessed with Annexin V-fluorescein isothiocyanate (V-FITC), and this was substantially enhanced
by 1400W or cPTIO, again consistent with iNOS/NO-imposed resistance . Thus, it appeared that
[56]
NOS-derived NO in U87 and U251 cells was acting cytoprotectively after a PDT-like challenge. When
immunoblot analysis was used to assess iNOS status in these cells, it was found that the enzyme level
increased progressively during post-irradiation incubation. After 6 h, it reached ~4-times the basal level
in U87 cells [Figure 2A] as well as U251 cells [Figure 2B]. As expected for this phenotype, U87 cells also
[56]
expressed nNOS , but, unlike iNOS, it was not upregulated after ALA/light treatment [Figure 2A].
Therefore, both glioblastoma cell types studied added significantly to their expressed iNOS the after a
photodynamic challenge, and the resulting NO clearly enhanced their resistance to photokilling. Evidence
for a large boost in NO steady state level was obtained by using the fluorescent probe diaminofluorescein-