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Page 2 of 15 Girotti et al. J Cancer Metastasis Treat 2020;6:52 I http://dx.doi.org/10.20517/2394-4722.2020.107

INTRODUCTION
Glioblastoma, also known as glioblastoma multiforme (GBM), is classified as a grade IV glioma by the
[1-3]
World Health Organization and is one of the most aggressive and persistent of all known human tumors .
The yearly incidence of glioblastoma in the United States is ~3 per 100,000 individuals. Difficulties in
distinguishing highly invasive malignant zones from normal brain tissue make tumor resection very
[2,3]
challenging . Glioblastomas are known to be resistant to most conventional interventions, including
[4-6]
ionizing radiation or chemotherapy with drugs such as cisplatin and temozolomide . Drug resistance
[7]
can either be inherent or acquired during treatment . Photodynamic therapy (PDT), which employs non-
ionizing radiation, has several advantages over radiotherapy or chemotherapy, including the ability to
often overcome resistance associated with these treatments [8-10] . Nevertheless, various forms of pre-existing
or treatment-induced resistance also apply for PDT [11,12] . One important example pertains to nitric oxide
(NO) generated by inducible nitric oxide synthase (iNOS) in PDT-challenged tumor cells. There is solid
evidence for this mode of resistance in glioblastoma cells as well as several other human cancer lines,
including breast, prostate, and melanoma [13,14] . In addition to this anti-PDT effect, iNOS/NO has been
shown to stimulate proliferation, migration, and invasion of cells that survive a photodynamic challenge.
In this review, we discuss findings such as these and their implications on anti-glioma PDT at the clinical
level. Relevant topics include: (1) NO and its underlying role in tumor promotion/persistence; (2) basic
principles of PDT and how it suppresses solid tumors; (3) iNOS/NO-mediated hyper-resistance to PDT
and hyper-aggressiveness of surviving cells; (4) mechanism of iNOS/NO induction by PDT; (5) tumor
expansion via PDT-induced bystander effects; and (6) pharmacologic approaches for limiting the negative
effects of iNOS/NO after PDT. Much of this discussion is based on studies carried out in the authors’
laboratories.

Two key aspects of these studies distinguish them from most others dealing with the pro-tumor effects of
iNOS/NO: (1) Rather than simply using unchallenged tumor cells, we applied an oxidative stress-based
challenge, viz. PDT, and assessed how it was affected by endogenous iNOS/NO; and (2) We discovered that,
in most cases, it was PDT-upregulated iNOS rather than pre-existing (constitutive) enzyme that generated
sufficient NO to stimulate resistance and surviving cell aggressiveness. Better recognition of these negative
responses to PDT is needed in advance of developing approaches for mitigating them and improving PDT
efficacy. What we discuss here may also provide new insights into how iNOS/NO could impact anti-tumor
chemotherapy or radiotherapy.

NITRIC OXIDE: TUMOR-PROMOTING VERSUS TUMOR-SUPPRESSING EFFECTS
NO is a short-lived free radical molecule (τ < 2 s in H O) that diffuses freely on its own in aqueous media
2
and, similar to O , can partition into hydrophobic environments such as cell membranes [15,16] . Naturally
2
occurring NO is generated by three enzyme isoforms in the nitric oxide synthase family: neuronal (nNOS/
NOS1), inducible (iNOS/NOS2), and endothelial (eNOS/NOS3) [17,18] . Whereas nNOS and eNOS operate
2+
at low constitutive levels and require Ca and calmodulin for optimal activity, iNOS can be induced to
2+
[18]
relatively high levels and does not require stimulatory Ca or calmodulin . All three enzymes catalyze
the five-electron oxidation of L-arginine to L-citrulline and NO at the expense of NADPH and O . NO is
2
involved in many different normo- and pathophysiologic processes. For example, eNOS-derived NO at
low steady state levels (1-10 nM) stimulates cyclic-GMP formation, leading to blood vessel relaxation and
lowering of blood pressure. In contrast, iNOS-derived NO at much higher levels (≥ 1 μM), as produced by
vascular macrophages in response to infection, is cytotoxic and potentially carcinogenic, e.g., by inducing
DNA mutations [19,20] . NO itself may act thusly by binding to iron in iron-sulfur or heme proteins, but often
−
does so after reacting with superoxide radical (O ) to give peroxynitrite (ONOO ), a strong indiscriminate
−
2
[21]
−
oxidant . If generated chronically, ONOO may be carcinogenic, e.g., by causing tyrosine nitration or
initiating lipid peroxidation . On the other hand, for established tumors, ONOO can be cytotoxic, since
[21]
−