Gliomas are aggressive brain tumors that are resistant to conventional chemotherapy and radiotherapy. Much of this resistance is attributed to endogenous nitric oxide (NO). Recent studies revealed that 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has advantages over conventional treatments for glioblastoma. In the present study, we used an in vitro model to assess whether NO from glioblastoma cells can interfere with ALA-PDT. Human U87 and U251 cells expressed significant basal levels of neuronal NO synthase (nNOS) and its inducible counterpart (iNOS). After an ALA/light challenge, iNOS level increased 3–4 fold over 24 h, whereas nNOS remained unchanged. Elevated iNOS resulted in a large increase in intracellular NO. Extent of ALA/light-induced apoptosis increased substantially when an iNOS inhibitor or NO scavenger was present, implying that iNOS/NO was acting cytoprotectively. Moreover, cells surviving a photochallenge exhibited a striking increase in proliferation, migration, and invasion rates, iNOS/NO again playing a dominant role. Also observed was a large iNOS/NO-dependent increase in matrix metalloproteinase-9 activity, decrease in tissue inhibitor of metalloproteinase-1 expression, and increase in survivin and S100A4 expression, each effect being consistent with accelerated migration/invasion as a prelude to metastasis. Our findings suggest introduction of iNOS inhibitors as pharmacologic adjuvants for glioblastoma PDT.

Employing an in vitro model for 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), we recently reported that human prostate cancer PC3 cells rapidly and persistently overexpressed inducible nitric oxide synthase (iNOS) and nitric oxide (NO) after a moderate ALA/light challenge. The upregulated iNOS/NO was shown to play a key role in cell resistance to apoptotic photokilling and also in the dramatic growth spurt observed in surviving cells. In the present study, we found that PC3 cells surviving an ALA/light insult not only proliferated faster than non-stressed controls, but migrated and invaded faster as well, these effects being abrogated by an iNOS inhibitor or NO scavenger. Photostressed prostate DU145 cells exhibited similar behavior. Using in-gel zymography, we showed that PC3 extracellular matrix metalloproteinase-9 (MMP-9) was strongly activated 24 h after ALA/light treatment and that MMP-9 inhibitor TIMP-1 was downregulated, consistent with MMP-9 involvement in enhanced invasiveness. We also observed a photostress-induced upregulation of α6 and β1 integrins, implying their involvement as well. The MMP-9, TIMP-1, and integrin effects were strongly attenuated by iNOS inhibition, confirming NO’s role in photostress-enhanced migration/invasion. This study reveals novel, potentially tumor-promoting, side-effects of prostate cancer PDT which may be averted through use of iNOS inhibitors as PDT adjuvants.

Many malignant tumors employ iNOS-derived NO to resist eradication by chemotherapeutic agents or ionizing radiation. In this study, we determined whether human breast carcinoma MDA-MB-231 cells in vitro and in vivo as tumor xenografts would exploit endogenous iNOS/NO to resist the cytotoxic effects of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT). Broad band visible irradiation of ALA-treated cells resulted in a marked after-light upregulation of iNOS protein which persisted for at least 24 h. Apoptotic killing of ALA/light-challenged cells was significantly enhanced by iNOS inhibitors (1400W, GW274150) and a NO trap (cPTIO), implying that stress-induced iNOS/NO was acting cytoprotectively. We found that cells surviving the photostress proliferated and migrated more rapidly than controls in 1400W- and cPTIO-inhibitable fashion, indicating iNOS/NO involvement. Female SCID mice bearing MDA-MB-231 tumors were used for animal model experiments. ALA-PDT with a 633 nm light source caused a significant reduction in post-irradiation tumor growth relative to light-only controls, which was further reduced by administration of 1400W or GW274150, whereas 1400W had little or no effect on controls. Immunoblot analyses of tumor samples revealed a progressive post-PDT upregulation of iNOS, which reached >5-times the control level after six days. Correspondingly, the nitrite/nitrate level in post-PDT tumor samples was substantially higher than that in controls. In addition, a 1400W-inhibitable upregulation of pro-survival/progression effector proteins such as Bcl-xL, Survivin, and S100A4 was observed after in vitro and in vivo ALA-PDT. This is the first known study to demonstrate iNOS/NO-induced resistance to PDT in an in vivo human tumor model.

Endogenous nitric oxide (NO) generated by inducible NO synthase (iNOS) promotes glioblastoma cell proliferation and invasion, and also plays a key role in glioblastoma resistance to chemotherapy and radiotherapy. Non-ionizing photodynamic therapy (PDT) has anti-tumor advantages over conventional glioblastoma therapies. Our previous studies revealed that glioblastoma U87 cells upregulate iNOS after a photodynamic challenge and that resulting NO not only increased resistance to apoptosis, but rendered surviving cells more proliferative and invasive. These findings were largely based on the effects of inhibiting iNOS activity and scavenging NO. Demonstrating now that iNOS expression in photostressed U87 cells is mediated by NF-κB, we hypothesized that (i) recognition of acetylated lysine (acK) on NF-κB p65/Rel A by bromodomain and extra-terminal (BET) protein Brd4 is crucial, and (ii) by suppressing iNOS expression, a BET inhibitor (JQ1) would attenuate the negative effects of photostress. The following evidence was obtained: (i) Like iNOS, Brd4 protein and p65-acK levels increased several fold in photostressed cells; (ii) JQ1 at minimally toxic concentrations had no effect on Brd4 or p65-acK upregulation after PDT, but strongly suppressed iNOS, survivin, and Bcl-xL upregulation, along with the growth and invasion spurt of PDTsurviving cells; (iii) JQ1 inhibition of NO production in photostressed cells closely paralleled that of growth/invasion inhibition; (iv) At 1% the concentration of iNOS inhibitor 1400W, JQ1 reduced post-PDT cell aggressiveness to a far greater extent. This is the first evidence for BET inhibitor targeting of iNOS expression in cancer cells and how such targeting can markedly improve therapeutic efficacy.

No abstract

Nitric oxide (NO) generated endogenously by inducible nitric oxide synthase (iNOS) promotes growth and migration/invasion of glioblastoma cells and also fosters resistance to chemotherapy and ionizing radiotherapy. Our recent studies revealed that glioblastoma cell iNOS/NO also opposes the cytotoxic effects of non-ionizing photodynamic therapy (PDT), and moreover stimulates growth/migration aggressiveness of surviving cells. These negative responses, which depended on PI3K/Akt/NF-κB activation, were strongly suppressed by blocking iNOS transcription with JQ1, a BET bromodomain inhibitor. In the present study, we sought to identify additional molecular events that precede iNOS transcriptional upregulation. Akt activation, iNOS induction, and viability loss in PDT-challenged glioblastoma U87 cells were all strongly inhibited by added L-histidine, consistent with primary involvement of photogenerated singlet oxygen (1 O 2). Transacetylase p300 not only underwent greater Akt-dependent activation after PDT, but greater interaction with NF-κB subunit p65, which in turn exhibited greater K310 acetylation. In addition, PDT promoted intramolecular disulfide formation and inactivation of tumor suppressor PTEN, thereby favoring Akt and p300 activation leading to iNOS upregulation. Importantly, deacetylase Sirt1 was down-regulated by PDT stress, consistent with the observed increase in p65-acK310 level, which fostered iNOS transcription. This study provides new mechanistic insights into how glioblastoma tumors can exploit iNOS/NO to not only resist PDT, but to attain a more aggressive survival phenotype.

Voltage-gated T-type

Many studies have shown that low flux nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in various tumors, including glioblastomas, can promote angiogenesis, cell proliferation, and migration/invasion. Minimally invasive, site-specific photodynamic therapy (PDT) is a highly promising anti-glioblastoma modality. Recent research in the authors’ laboratory has revealed that iNOS-derived NO in glioblastoma cells elicits resistance to 5-aminolevulinic acid (ALA)-based PDT, and moreover endows PDT-surviving cells with greater proliferation and migration/invasion aggressiveness. In this contribution, we discuss iNOS/NO antagonism to glioblastoma PDT and how this can be overcome by judicious use of pharmacologic inhibitors of iNOS activity or transcription.