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 up-regulate iNOS after a photodynamic challenge and that the resulting NO not only increases resistance to apoptosis but renders 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/RelA 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 severalfold in photostressed cells. (ii) JQ1 at minimally toxic concentrations had no effect on Brd4 or p65-acK up-regulation after PDT but strongly suppressed iNOS, survivin, and Bcl-xL up-regulation, along with the growth and invasion spurt of PDT-surviving cells. (iii) JQ1 inhibition of NO production in photostressed cells closely paralleled that of growth/invasion inhibition. (iv) Finally, 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.
Highlights
Most established malignant tumors exist under moderate inflammatory conditions, which foster tumor cell survival, proliferation, and metastatic expansion [1,2,3]
We tested the sensitivity of glioblastoma U87 cells to the bromodomain and extra-terminal domain (BET) bromodomain inhibitor JQ1 and compared this with cell sensitivity to photodynamic therapy (PDT) and PDT combined with JQ1
Cytotoxicity was examined as extent of apoptotic cell death, which for our PDT approach occurs via the intrinsic pathway, because protoporphyrin IX (PpIX) is localized mainly in mitochondria [15, 26]
Summary
We tested the sensitivity of glioblastoma U87 cells to the BET bromodomain inhibitor JQ1 and compared this with cell sensitivity to PDT and PDT combined with JQ1. The p65 immunoprecipitation revealed a strong Brd immunoblot band, the intensity of which was substantially reduced by JQ1 We deduced from this evidence that Brd served as a co-activator of NF-B/p65 in photostressed cells and that JQ1 suppressed iNOS induction by targeting Brd and preventing its binding to p65-acK310. Bay, which strongly reduced iNOS expression via inhibition of NF-B activation (Fig. 4), suppressed post-PDT invasiveness to nearly the same extent as JQ1. The striking decline in c-Myc and return to constitutive level after 24 h (Fig. 10E) might reflect a unique stress accommodation response of this effector in preparation for accelerated cell division We concluded from these findings that PDT stressinduced iNOS played a major role in promoting cell resistance and aggressiveness, the altered expression of survivin, Bcl-xL, p21, MMP-9, and c-Myc made a significant contribution, which could be significantly counteracted by inhibition of BET bromodomains by JQ1
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