NO‐sulindac inhibits the hypoxia response of PC‐3 prostate cancer cells via the Akt signalling pathway

Abstract

Nitric oxide-donating non-steroidal anti-inflammatory drugs are safer than traditional NSAIDs and inhibit the growth of prostate cancer cells with greater potency than NSAIDs. In vivo, prostate cancer deposits are found in a hypoxic environment which induces resistance to chemotherapy. The aim of this study was to assess the effects and mechanism of action of a NO-NSAID called NOsulindac on the PC-3 prostate cancer cell line under hypoxic conditions. NO-sulindac was found to have pro-apoptotic, cytotoxic, and anti-invasive effect on PC-3 cells under normoxia and hypoxia. NO-sulindac was significantly more cytotoxic than sulindac at all oxygen levels. The sulindac/linker and NO-releasing subunits both contributed to the cytotoxic effects of NO-sulindac. Resistance of PC-3 cells to NO-sulindac was induced as the oxygen concentration declined. Hypoxia-induced chemoresistance was reversed by knocking-down hypoxia-inducible factor-1a (HIF-1a) mRNA using RNAi. Nuclear HIF-1a levels were upregulated at 0.2% oxygen but reduced by treatment with NO-sulindac, as was Akt phosphorylation. NO-sulindac treatment of hypoxic PC-3 cells transfected with a reporter construct, downregulated activation of the hypoxia response element (HRE) promoter. Co-transfection of PC-3 cells with the HRE promoter reporter construct and myrAkt (constitutively active Akt) plasmids reversed the NO-sulindac induced reduction in HRE activation. Real-time polymerase chain reaction analysis of hypoxic, NO-sulindac treated PC-3 cells showed downregulation of lysyl oxidase and carbonic anhydrase IX mRNA expression. Collectively, these novel findings demonstrate that NO-sulindac directly inhibits the hypoxia response of PC-3 prostate cancer cells by inhibiting HIF-1a translation via the Akt signalling pathway. The ability of NO-sulindac to inhibit tumour adaption to hypoxia has considerable relevance to the future management of prostate cancer with the same cellular properties as PC-3. ' 2008 Wiley-Liss, Inc.Key words: hypoxia; prostate cancer; nitric oxide donors; NONSAIDs; AktIn Western Europe and North America, prostate cancer is the commonest cancer and the second most common cause of cancer death in men. In the United Kingdom, prostate cancer accounts for 23% of all new male cancer diagnoses and 13% of male cancerrelated deaths. 1 Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit tumourigenesis in a variety of cancers. [2][3][4] In vitro studies have shown that conventional NSAIDs, at physiological achievable doses, inhibit the proliferation of immortalised human prostate cancer cells. 5 Additionally, meta-analyses of observational studies of men taking regular NSAIDs have reported statistically significant reductions in risk of prostate cancer. 6,7 However, conventional NSAIDs have several side-effects, particularly gastrointestinal bleeding, which limit their use in elderly patients with prostate cancer. In an attempt to reduce the side-effects of traditional NSAIDs, cyclooxygenase-2 inhibitors and nitric oxide (NO • )-donating non-steroidal...

DNA strand breaks and hypoxia response inhibition mediate the radiosensitisation effect of nitric oxide donors on prostate cancer under varying oxygen conditions

“…This work complements and expands our previous studies which demonstrated the cytotoxic effects of NO-NSAIDs on prostate cancer cells under normoxia and hypoxia [6,7]. The use of both 2D and 3D models showed that NO-sulindac radiosensitised the PC-3 hormone insensitive prostate cancer cell line, not only under normoxia but particularly under chronic hypoxia.…”

“…4A). As we have shown previously, treatment of PC-3 cells with 25μM NO-sulindac under both normoxia and hypoxia reduces the levels of HIF-1α [7]. Furthermore, HIF-1α levels were undetectable on western blotting following NO-sulindac and radiation treatment at 8, 24 or 48 hours post-irradiation (Fig.…”

“…HIF-1 is the transcription factor acting as the master regulator of the hypoxia response; the HIF-1α component is most important in this regulatory process [1]. Previously we have shown that NONSAIDs were cytotoxic to prostate cancer cell lines under both normoxia and hypoxia and causes a post-transcriptional reduction in HIF-1α nuclear protein [7]. The data presented in the current study demonstrates the effect that irradiation combined with NO-sulindac exposure inhibited the hypoxia response (using HIF-1α production as a 17 surrogate).…”

“…Western blots for hypoxia inducible factor (HIF)-1α were performed on nuclear protein extracts as described previously [7,15].…”

“…We and others have demonstrated that NO-NSAIDs prevent the development of malignancy and are powerful agents against established cancer deposits in vitro and in vivo [4][5][6]. Recently we have determined the function of NO-sulindac under hypoxic conditions and shown that this agent inhibits the hypoxia response in the PC-3 prostate cancer cell line via the Akt pathway [7]. Independently, NO and NSAIDs have both been shown to behave as radiosensitising agents [8,9].…”