Upstream signaling events leading to elevated production of pro-survival nitric oxide in photodynamically-challenged glioblastoma cells

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.

Rosmarinic Acid Induces Proliferation Suppression of Hepatoma Cells Associated with NF-κB Signaling Pathway

“…These results are similar to the reported role of RA in colorectal cancer [33]. Numerous studies have shown that the PI3K/Akt/NF-κB pathway is closely related to HCC progression [34][35][36]. In line with these reports, our results showed that the PI3K/Akt/NF-κB signal pathway is involved in the anti-tumor effects of RA in HCC.…”

Nitric oxide-elicited resistance to anti-glioblastoma photodynamic therapy

“…Based on non-glioma studies by Huang et al [49] , it was postulated that specific lysine acetylation on p65 is necessary for stimulating transcriptional activity. Fahey and Girotti [48] verified this by showing that acetylation of lysine-310 (p65-acK310) is substantially upregulated in photostressed U87 cells. Investigation of upstream signaling events revealed that p65-acK310 formation was dependent on phosphorylation-activation of PI3K, followed by Akt, and thence acetyltransferase p300.…”

“…For mechanistic understanding, most efforts to date have focused on how iNOS/NO is upregulated rather than how NO signals for greater cell resistance or aggressiveness, although some progress has been made in the latter category. Working with U87 and U251 cells, Fahey and Girotti [48] found that transcription factor NF-κB played a seminal role in post-ALA/light iNOS induction leading to greater cell migration and invasion. Consistent with this, NF-κB subunit p65/Rel A of photostressed U87 cells translocated from cytosol to nucleus, where it participated in iNOS transcription [48] .…”

“…Working with U87 and U251 cells, Fahey and Girotti [48] found that transcription factor NF-κB played a seminal role in post-ALA/light iNOS induction leading to greater cell migration and invasion. Consistent with this, NF-κB subunit p65/Rel A of photostressed U87 cells translocated from cytosol to nucleus, where it participated in iNOS transcription [48] . Based on non-glioma studies by Huang et al [49] , it was postulated that specific lysine acetylation on p65 is necessary for stimulating transcriptional activity.…”

“…Investigation of upstream signaling events revealed that p65-acK310 formation was dependent on phosphorylation-activation of PI3K, followed by Akt, and thence acetyltransferase p300. These sequential activations were supported (at least in part) by inactivation of tumor-suppressor PTEN (PIP 3 phosphatase) via intramolecular -S-S-bond formation [48] . The rise in acK310 level after photostress was suppressed by an inhibitor of activated p300, confirming that the latter had catalyzed acetylation of p65-K310 [48] .…”