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Page 12 of 15 Girotti et al. J Cancer Metastasis Treat 2020;6:52 I http://dx.doi.org/10.20517/2394-4722.2020.107
[83]
promoter sites . BET protein inhibitors such as JQ1 and OTX015 were recently introduced as powerful
new means of suppressing tumor development and progression at the transcriptional level [84,85] . These
inhibitors function by binding to BET domains on Brd4 and other BET proteins, thereby preventing
[85]
interaction with acK groups on transcription factors (e.g., p65-acK310) or on histones . When tested
on ALA/light-treated U87 cells, JQ1 at a minimally cytotoxic concentration: (1) increased cell killing
synergistically compared with photostress alone; (2) strongly inhibited Brd4 binding to p65-acK310; (3)
greatly reduced iNOS/NO upregulation after irradiation; and (4) nearly abolished the hyper-aggressiveness
of cells that could withstand the ALA/light challenge . One other striking observation in this study is
[63]
that the concentration of JQ1 used (~0.3 μM) was far below that of 1400W capable of producing similar
effects. Another glioblastoma line, U251 cells, responded similarly to JQ1 after being photostressed .
[63]
In addition to iNOS, several other NF-κB-regulated proteins were affected by photostress in U87 cells,
including pro-survival Bcl-xL and Survivin, which were upregulated, and tumor suppressor p21, which
[63]
was downregulated . Each of these photostress responses, similar to iNOS upregulation, was strongly
[63]
suppressed by JQ1, thereby promoting cell photokilling . Although Bcl-xL and Survivin transcription
may have been directly affected by JQ1, an indirect iNOS/NO-mediated effect was also possible, since NO
is known to modulate expression of these effector proteins [19,20] . Thus, in at least these two cases, JQ1 could
have acted directly by preventing Brd4 binding at promoter sites and/or indirectly by inhibiting iNOS
expression. In any event, JQ1 inhibition of iNOS transcription appeared to play the major role in improving
the efficacy of glioblastoma cell photokilling. It is clear, therefore, that JQ1 would make a highly promising
PDT adjuvant, particularly since it has already been used successfully with other anti-cancer therapies.
In the case of glioblastoma, for example, JQ1 has been reported to synergize with temozolomide in
cytotoxicity at the in vitro as well as in vivo level . We anticipate that JQ1 or some other BET inhibitor will
[86]
act similarly when used in combination with PDT in glioma animal models and eventually glioma patients.

CONCLUSION
The many attractive features of ALA-PDT, including tumor site specificity, non-toxicity of components
individually (ALA-induced PpIX, light, and O ), and its demonstrated efficacy on difficult tumors such
2
as glioblastomas, make it an appealing therapy for these malignancies [8-10,41] . An added advantage of using
ALA is that tumor-localized PpIX can be employed for fluorescence-guided surgery (FGS), which is often
[43]
followed up by PDT to eradicate any residual cells . It is now well established that cells in many tumors,
including gliomas, exploit low-level NO to avoid apoptosis, stimulate proliferation and migration, and
resist radio- or chemotherapy [19-21] . As pointed out above, such NO can also impose a strong resistance to
PDT. The NO can derive from tumor cells themselves, although proximal vascular cells (macrophages,
fibroblasts, and endothelial cells) may contribute. The in vitro and in vivo studies described in this review
are unique in demonstrating that endogenous iNOS/NO in many tumor cells, including glioblastomas,
plays a major role not only in PDT resistance, but also enhanced aggressiveness of surviving cells and non-
targeted bystanders. Although both basal and photostress-induced iNOS might be implicated in these
responses, there is now solid evidence that induced enzyme plays a preponderant role in several cancer
[13]
types . This evidence is unprecedented because most therapy-based studies up to now have considered
only pre-existing iNOS/NO and not the possibility of overexpression due to the treatment itself. Concerns
about a more aggressive (proliferative and migratory/invasive) phenotype of PDT-surviving cells could be
mitigated by turning to pharmacologic inhibitors of iNOS enzymatic activity or iNOS transcription. We
suggest possible candidates in each of these categories, emphasizing the greater advantages of those in the
latter category, i.e., BET inhibitors.

DECLARATIONS
Acknowledgements
Reshma Bhowmick, Magda Niziolek, Mariusz Zareba, Anna Pilat, and Jerzy Bazak are thanked for their
valuable contributions to the some of the studies described. Lisa Litzenberger is thanked for helping to
format and finalize the five figures.