Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1

ABSTRACT The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1␣ protein that acts as a dominant-negative HIF-1␣ (HIF-1␣-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1␣ was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1␣-no-TAD or nontoxic concentrations (0.1 M; ϽIC 10 ) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (Ͻ0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC 50 values in anoxia were 3-fold higher than those in air for HT1080 (2.2 Ϯ 0.3 versus 0.7 Ϯ 0.2 M) and HCT116 (9 Ϯ 4 versus 3 Ϯ 2 M) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC 50 in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1␣-no-TAD (multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC 50 values: HT1080, 0.7 Ϯ 0.04 M; HCT116, 3 Ϯ 1 M). HIF-1␣-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure

Results of endoscopic transsphenoidal pituitary surgery in 40 patients with a growth hormone-secreting macroadenoma

Contains fulltext : 96290.pdf (Publisher’s version ) (Open Access)OBJECTIVE: Transsphenoidal pituitary surgery (TS) is the primary treatment of choice for patients with acromegaly. Macroadenomas (>1 cm) are more difficult to resect than microadenomas (remission rate +/- 50% compared to +/- 90%). Besides the conventional microscopic TS, the more recently introduced endoscopic technique is nowadays frequently used. However, no large series reporting on its results have yet been published. We evaluated the outcome of endoscopic TS in 40 patients with a growth hormone (GH)-secreting macroadenoma treated in our hospital between 1998 and 2007. METHODS: Medical records were retrospectively reviewed. Remission was defined as disappearance of clinical symptoms of acromegaly, normal serum insulin-like growth factor-1 levels (</=2 SD) and serum GH levels suppressed to <2 mU/l after an oral glucose tolerance test within the first 4 months after TS. RESULTS: In four patients TS aimed at debulking of the tumour. In the remaining 36 patients, remission was achieved in 20 patients. In the first 5 years remission was achieved in 6 out of 18 patients (33%) compared to 14 out of 22 patients (63%) in the following 5 years (p = 0.06). Thirteen patients had a mild perioperative complication. Before TS 15 patients received hormonal substitution therapy compared to 12 patients (33%) after TS. CONCLUSION: Endoscopic TS is a good primary therapeutic option for patients with a GH-secreting macroadenoma, resulting in a remission rate of up to 63% in experienced hands. This technique can potentially improve the outcome of TS in these patients

Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1

ABSTRACT The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1␣ protein that acts as a dominant-negative HIF-1␣ (HIF-1␣-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1␣ was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1␣-no-TAD or nontoxic concentrations (0.1 M; ϽIC 10 ) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (Ͻ0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC 50 values in anoxia were 3-fold higher than those in air for HT1080 (2.2 Ϯ 0.3 versus 0.7 Ϯ 0.2 M) and HCT116 (9 Ϯ 4 versus 3 Ϯ 2 M) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC 50 in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1␣-no-TAD (multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC 50 values: HT1080, 0.7 Ϯ 0.04 M; HCT116, 3 Ϯ 1 M). HIF-1␣-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure

MOL 15743 1 TITLE PAGE REVERSING HYPOXIC CELL CHEMORESISTANCE IN VITRO USING GENETIC AND SMALL MOLECULE APPROACHES TARGETING HYPOXIA INDUCIBLE FACTOR-1 MOL 15743 2 RUNNING TITLE PAGE Running Title: HIF-1 INHIBITION AND HYPOXIC CHEMOSENSITISATION Correspon

ABSTRACT The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenoviral-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumour cells to etoposide. The genetic therapy exploited a truncated HIF-1α protein that acts as a dominant negative (HIF-1α-no-TAD). Its functionality was validated in six human tumour cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant negative was nuclear localised and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analogue DX-52-1 and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1α-no-TAD or non-toxic (0.1µM; &lt;IC 10 ) concentrations of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (&lt;0.01% oxygen). Topotecan only inhibited HIF-1 activity at cytotoxic concentrations and was not used in the combination study. Etoposide IC 50 values in anoxia were three-fold higher than those in air for HT1080 (2.2±0.3 versus 0.7±0.2µM) and HCT116 (9±4 versus 3±2µM) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC 50 in HT1080 cells whilst only KW2152 yielded sensitisation in HCT116 cells. In contrast AdHIF-1α-no-TAD (MOI 50) ablated the anoxic resistance in both cell lines (IC 50 values: HT1080, 0.7±0.04µM; HCT116, 3±1µM). HIF-1α-no-TAD expression inhibited HIF-1-mediated down-regulation of the pro-apoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy where hypoxic cell resistance contributes to treatment failure. MOL 15743 4 The resistance of hypoxic cells to anti-cancer therapeutic strategies has a profound impact on treatment response. Within solid tumours hypoxic cell chemoresistance was originally attributed to poor drug distributions and to the contention that hypoxic tumour cells are predominantly quiescent. Pre-clinical observations have additionally demonstrated that hypoxia is a selective pressure for genomic instability with acquired drug resistance and loss of apoptotic potential reported for cells exposed to hypoxia both in vitro and in vivo MATERIALS AND METHODS Vector construction. The method for generating pcDNA3.1/Zeo expressing a truncated HIF-1α construct lacking the transactivation domains (TAD) was based on a published strategy (Jiang 1996). The TAD was removed from the HIF-1α cDNA (contained in pBSSKII+) using Not1/AflII and a linker inserted (5&apos;-TTAAGTGAGCTTTTTCTTAATCTAGAGC-3&apos;). HIF-1α-no-TAD (1.1kb fragment) was then isolated (Not1/Kpn1) and cloned into the pcDNA3.1/Zeo vector (Invitrogen, UK) generating pHIF-1α-no-TAD. To construct pHIF-1α-no-TAD-EGFP, the sequence encoding EGFP was removed from pIRES2EGFP (BD Biosciences Clontech, UK) and introduced upstream and in frame of HIF-1α-no-TAD in pBSSKII+. HIF-1α-no-TAD-EGFP was isolated by Not1/Kpn1 digestion and cloned into pcDNA3.1/Zeo to generate pHIF-1α-no-TAD-EGFP. Adenoviral vectors. AdHIF-1α-no-TAD was made using the pAd Easy system (Stratagene, USA). HIF-1α-no-TAD was isolated and cloned into a modified pShuttle containing the elongation factor-1α (EF-1) promoter. pShuttle-HIF-1α-no-TAD and pAdEasy were cotransformed into BJ5183 competent cells (Stratagene, UK) generating pAdHIF-1α-no-TAD. Primary viral inoculums and large-scale preparations were generated in human embryonic promoter. The Carbonic Anhydrase -9 reporter has been described previously (Wycoff 2000) and consisted of the sequence -506/+43 in the native gene cloned into pGL3-basic (Promega, UK). Cell lines. All of the human tumour cell lines which included HT1080 (fibrosarcoma), DU145 (prostate carcinoma), U87 (glioma), T47D (breast carcinoma), HCT116 and HT29 (colon carcinoma) and the rodent lines were cultured in RPMI (Gibco BRL, UK) containing 10% foetal calf serum and 2mM glutamine in a 95% air: 5% CO 2 environment. All were free from mycoplasma contamination (Mycotect; Gibco BRL, UK). The CHO-K1 derived HREreporter strain, C4.5, expresses human CD-2 regulated by the murine PGK-1-HRE sequence. Ka13.5 cells were derived from C4.5 and are HIF-1α-deficient (Wood 1998). The HCT116 HRE-reporter strain expresses the LDH-A SV40 min driven Firefly luc+ cassette detailed above encoded within pCI-neo (Promega, UK). Exposure to anoxia or the hypoxic mimetic cobalt chloride (100µΜ) for 16h yields a robust (~10 fold) induction of luciferase activity in these cells (Williams and Cowen, unpublished observations). The Hepa-1 wt and HIF-1β-deficient derivative Hepa-1 c4 are murine hepatoma cells and have been extensively described (Maxwell 1997; Ryan 1998; Griffiths 2002; Williams 2002 Assays were performed on ice using 0.01mg ml -1 lyophilised Carbonic Anhydrase RESULTS Subcellular localisation and oxygen dependence of HIF-1α-no-TAD. To generate the HIF- 1 dominant negative HIF-1α-no-TAD the transactivation domains (TAD) were removed from the coding sequence of human HIF-1α. This deleted the oxygen dependent degradation domain (ODDD) whilst leaving those regions required for dimerisation and DNA binding intact Transfection with the control vector pIRESEGFP resulted in EGFP expression that was confined to the cytoplasm Functionality of HIF-1α-no-TAD. The ability of HIF-1α-no-TAD to interfere with HIF-1 mediated gene expression was evaluated against a luciferase reporter construct comprising a trimer of the HIF-1 recognition sequence (hypoxia-responsive element; HRE) from the lactate dehydrogenase (LDH) promoter. A range of tumour cell lines were transiently transfected with the LDH-HRE reporter construct alone or in combination with pHIF-1α-no-TAD. The dominant negative decreased HRE-mediated luciferase expression in all cell lines in anoxia T47D and HT1080 cells and demonstrated that HIF-1α-no-TAD could similarly inhibit MOL 15743 11 expression driven by the HREs from the PGK (phosphoglycerate kinase) and carbonic anhydrase-9 genes in both air and anoxia promoter context, HIF-1α no TAD had no significant effect on gene expression regulated by the constitutive human elongation factor-1 (EF-1) promoter in the cell line panel (data not shown). Validation of an adenoviral vector expressing HIF-1α-no-TAD. HIF-1α-no-TAD was cloned into a replication deficient type 5-adenovirus vector to generate AdHIF-1α-no-TAD. Adenoviral infection of HCT116 cells that stably express the LDH-HRE-luciferase cassette ( This was not the case for topotecan where 50% HIF-1 inhibition was only achieved using a concentration 12 fold higher (600nM) than the IC 50 (0.05±0.04µM). Furthermore the concentration of topotecan required to inhibit constitutive, EF-1 driven reporter expression by 50% was lower than that required for HRE inhibition (100nM). This was not the case for DX-52-1 and KW2152 with 50% inhibition of EF-1-mediated expression achieved using concentrations of 900nM and 800nM respectively. AdHIF-1α-no-TAD and non-toxic doses of DX-52-1 and KW2152 can reverse the anoxic resistance of tumour cells to etoposide. Preliminary studies were undertaken using Hepa-1 wt and the HIF-1β deficient Hepa-1 c4 to establish that HIF-1 function affects etoposide sensitivity. A two-fold increase in etoposide concentration was required to give the same level of cell kill in anoxic conditions compared with aerobic exposure in the Hepa-1 wt cells. The HIF-1 deficient Hepa-1 c4 cells, however, were more sensitive to etoposide in anoxia Again the anoxic drug resistance could not be related to cell cycle characteristics that were unaffected by the 16h anoxic exposure used were dosed at 100nM in combination with etoposide treatment. The direct cytotoxicity of topotecan precluded its use in the combination studies. In HT1080 cells, co-treatment with either drug significantly reduced the IC 50 for etoposide when exposed under anoxic conditions whilst having no significant effect on the aerobic IC 50 value We have recently elucidated the pro-apoptotic protein Bid as a target for HIF-1 mediated down-regulation under anoxic conditions (Erler 2004). Therefore we evaluated the impact of the dominant negative HIF-1α and DX-52-1 on the expression of Bid. Consistently HIF-1α-no-TAD reversed the anoxic down-regulation of Bid observed in untreated or empty vector control samples of both HT1080 and HCT116 cell