Nitric oxide antagonism to glioblastoma photodynamic therapy and mitigation thereof by BET bromodomain inhibitor JQ1

Abstract

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.

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

“…The possibility that PDT-enhanced acetylation of p65-K310, as observed previously [34], is facilitated by greater physical interaction of p300 with NF-κB/p65 was examined using an immunoprecipitation approach. After ALA/light treatment, U87 cells were switched to 10% FBS-medium and returned to the incubator for 6 h, after which they were lysed and total lysate protein determined.…”

“…Invasive cells that had traversed were detached by centrifugation into 10% FBS-medium, transferred to a 96-well plate, and quantified by CCK assay. Other details were as described previously [31][32][33][34].…”

“…We showed recently that iNOS transcription in glioblastoma cells is NF-κB-dependent and mediated by interaction of bromodomain protein Brd4 with acetylated K310 on the p65 subunit of NF-κB [34]. Blocking iNOS transcription with a Brd4-binding BET bromodomain inhibitor (JQ1) was found to be much more effective in limiting NO's anti-PDT effects than using inhibitors of iNOS enzymatic activity [34].…”

Targeting iNOS As a Valuable Strategy for the Therapy of Glioma

“…The BET family proteins have been identified in oncogenic rearrangements, and contribute to the development of different neoplastic diseases. Recently, it was found that JQ1 suppresses iNOS expression via Brd4 inhibition, enhancing the cytotoxic effects of photodynamic therapy on glioblastoma U87 cells . Indeed, a major reason of resistance to PDT therapy in glioblastomas, is that the PDT oxidative cellular damage induces the sustained up‐regulation of pro‐survival iNOS, and Brd4 was found as the predominant co‐activator for iNOS expression.…”

“…Recently, it was found that JQ1 suppresses iNOS expression via Brd4 inhibition, enhancing the cytotoxic effects of photodynamic therapy on glioblastoma U87 cells. [67] Indeed, a major reason of resistance to PDT therapy in glioblastomas, is that the PDT oxidative cellular damage induces the sustained up-regulation of pro-survival iNOS, and Brd4 was found as the predominant co-activator for iNOS expression. Therefore, JQ1 suppressed glioblastoma cells survival and invasiveness by the inhibition of the Brd4-Nf-kB-iNOS pathway.…”