The action of ß-hydroxybutyrate on the growth, metabolism and global histone H3 acetylation of spontaneous mouse mammary tumours: evidence of a ß-hydroxybutyrate paradox.

BACKGROUND: Ketone bodies have both metabolic and epigenetic roles in cancer. In several studies, they showed an anti-cancer effect via inhibition of histone deacetylases; however, other studies observed faster tumour growth. The related molecule butyrate also inhibits growth of some cancer cells and accelerates it in others. This "butyrate paradox" is thought to be due to butyrate mediating histone acetylation and thus inhibiting cell proliferation in cancers that preferentially utilise glucose (the Warburg effect); whereas in cells that oxidise butyrate as a fuel, it fails to reach inhibitory concentrations and can stimulate growth. METHODS: We treated transgenic mice bearing spontaneous MMTV-NEU-NT mammary tumours with the ketone body ß-hydroxybutyrate (ß-OHB) and monitored tumour growth, metabolite concentrations and histone acetylation. In a cell line derived from these tumours, we also measured uptake of ß-OHB and glucose, and lactate production, in the absence and presence of ß-OHB. RESULTS: ß-OHB administration accelerated growth of MMTV-NEU-NT tumours, and their metabolic profile showed significant increases in ATP, glutamine, serine and choline-related metabolites. The ß-OHB concentration within the treated tumours, 0.46 ± 0.05 µmol/g, had no effect on histone acetylation as shown by western blots. Cultured tumour cells incubated with 0.5 mM ß-OHB showed ß-OHB uptake that would be equivalent to 54% of glycolytic ATP phosphorylation and no significant change in glucose consumption or lactate production. CONCLUSIONS: These results suggest that a ß-OHB paradox may occur in these mammary tumours in a manner analogous to the butyrate paradox. At low ß-OHB concentrations (<1 mM, as observed in our tumour model post-treatment), and in the absence of a Warburg effect, ß-OHB is consumed and thus acts as an oxidative energy source and not as an epigenetic factor. This would explain the increase in tumour growth after treatment, the metabolic profiles and the absence of an effect on histone H3 acetylation.

Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy.

BACKGROUND: The importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours. METHODS: Mammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used. RESULTS: The NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells. CONCLUSIONS: The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.

In vivo 31P magnetic resonance spectroscopy using a needle microcoil.

Batch fabrication methods have been used to produce low-cost microcoils for magnetic resonance spectroscopy (MRS) that could be discarded after applications such as insertion into tissue during interventional surgery. Needle-shaped microcoils were constructed using electroplated conductors buried in shafts formed with different combinations of silicon and plastic and used to acquire in vivo (31)P spectra of rat thigh muscle at 81 MHz. The designs in this study achieved a maximum signal-to-noise ratio (SNR) for phosphocreatine (PCr) of 10.4 in a 10-min acquisition, with the three adenosine triphosphate (ATP) multiplets also clearly visible. An average 20% reduction in PCr occurred over a 60-min period, and intracellular pH was estimated to be 6.6, which are both evidence of ischemia. A needle microcoil design could have applications in real-time MRS of tumors or in evaluating pathology in general during surgical investigations.

In vivo determination of tumor oxygenation during growth and in response to carbogen breathing using 15C5-loaded alginate capsules as fluorine-19 magnetic resonance imaging oxygen sensors.

PURPOSE: The objective was to present a method for the repeated noninvasive measurement of tumor oxygenation (Po(2)) over the whole period of tumor growth. METHODS AND MATERIALS: A mixture of tumor homogenate (GH3 prolactinoma) and alginate capsules loaded with perfluoro-15-crown-5-ether (15C5) was injected into the flanks of Wistar Furth rats. The temporal behavior of tumor Po(2) was monitored between Day 1 and 26 after injection using fluorine-19 ((19)F) magnetic resonance imaging (MRI). In addition, the response of tumor Po(2) to modifiers of the tumor microenvironment (carbogen [95% O(2)/5% CO(2)], nicotinamide, and hydralazine) was investigated. RESULTS: An initial increase of tumor Po(2), probably reflecting neovascularization, followed by a decrease after Week 2, probably indicating tumor hypoxia or necrosis, were observed. The minimum and maximum average Po(2) +/- SEM observed were 3.3 +/- 2.0 mm Hg on Day 2 and 25.7 +/- 3.8 mm Hg on Day 13, respectively. Carbogen increased the tumor Po(2), whereas nicotinamide caused no significant change and hydralazine induced a significant decrease in tumor oxygenation. CONCLUSIONS: A preclinical method for the repeated noninvasive determination of tumor Po(2) was presented. It might help to investigate tumor physiology and the mechanisms of modifiers of the tumor microenvironment and their role in different therapeutic approaches.

Tumor R2* is a prognostic indicator of acute radiotherapeutic response in rodent tumors.

PURPOSE: To test the prognostic potential of tumor R2* with respect to radiotherapeutic outcome. Blood oxygenation level dependent (BOLD) MRI images are sensitive to changes in deoxyhemoglobin concentration through the transverse MRI relaxation rate R2* of tissue water, hence the quantitative measurement of tumor R2* may be related to tissue oxygenation. METHODS AND MATERIALS: Tumor growth inhibition in response to radiation was established for both GH3 prolactinomas and RIF-1 fibrosarcomas with animals breathing either air or carbogen during radiation. In a separate cohort, the baseline R2* and carbogen (95% O2, 5% CO2)-induced DeltaR2* of rat GH3 prolactinomas and murine RIF-1 fibrosarcomas were quantified using multigradient echo (MGRE) MRI prior to radiotherapy, and correlated with subsequent tumor growth inhibition in response to ionizing radiation, while the animals breathed air. RESULTS: A radiation dose of 15 Gy caused pronounced growth delay in both tumor models and transient regression of the GH3 prolactinomas. When the animals breathed carbogen during radiation, the growth delay/regression was enhanced only in the GH3 prolactinomas. The GH3 prolactinomas, which exhibit a relatively fast baseline R2* and large DeltaR2* in response to carbogen breathing prior to radiotherapy, showed a substantial reduction in normalized tumor volume to 66 +/- 3% with air breathing and 36 +/- 5% with carbogen seven days after 15 Gy irradiation. In contrast, the effect of 15 Gy on the RIF-1 fibrosarcomas, which give a relatively slow baseline R2* and negligible DeltaR2* response to carbogen prior to treatment, showed a much smaller growth inhibition (143 +/- 3% with air, 133 +/- 12% with carbogen). CONCLUSION: Quantitation of tumor R2* and carbogen-induced DeltaR2* by MGRE MRI provides completely noninvasive prognostic indicators of a potential acute radiotherapeutic response.

Issues in GRE & SE magnetic resonance imaging to probe tumor oxygenation.

Tumor oxygenation determines the efficacy of radiotherapy, but there is no non-invasive way to image this parameter. Since gradient recalled echo (GRE) images are sensitive to blood deoxyhaemoglobin concentration ([dHb]) they could have a role in assessing tumor oxygenation. In brain, linear relationships have been demonstrated between brain tissue R2* relaxation rate and tissue [dHb] or oxygen saturation, but in tumors, vascular and tissue heterogeneity, and the presence of simultaneous oxidative and glycolytic metabolism, complicate the analysis. We have studied the effects of vascular challenge in a rat prolactinoma tumor model by MR imaging and spectroscopy and comment on the implications of these results for calibrating GRE images for blood or tissue pO2.

Enhanced uptake of ifosfamide into GH3 prolactinomas with hypercapnic hyperoxic gases monitored in vivo by (31)P MRS.

Previously, (31)P magnetic resonance spectroscopy (MRS) has been used to detect ifosfamide (IF) in vivo and to show that breathing carbogen (5% CO(2)/95% O(2)) enhances the uptake and increases the efficacy of IF in rat GH3 prolactinomas [Rodrigues LM, Maxwell RJ, McSheehy PMJ, Pinkerton CR, Robinson SP, Stubbs M, and Griffiths JR (1997). In vivo detection of ifosfamide by (31)P MRS in rat tumours; increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas. Br J Cancer 75, 62-68]. We now show that other hypercapnic and/or hyperoxic (5% CO(2) in air, 2.5% CO(2) in O(2)) gas mixtures also increase the uptake of IF into tumors, measured by (31)P MRS. All gases caused an increased uptake (C(max)) of IF compared to air breathing, with carbogen inducing the largest increase (85% (P<.02) compared to 46% with 2.5% CO(2) in O(2) (P<.004) and 48% with 5% CO(2) in air (P<.004)). The T(max) (time of maximum concentration in tumor posintravenous injection of IF) was significantly (P<.04) later in the cohort that breathed 5% CO(2) in air. The increased uptake of IF with carbogen breathing was selective to tumor tissue and there were no significant increases in any of the normal tissues studied, suggesting that any host tissue toxicity would be minimal. Carbogen breathing by patients causes breathlessness. There was no significant difference in IF uptake between breathing carbogen and 2.5% CO(2) in O(2) and, therefore, the ability of 2.5% CO(2) in O(2) to also increase IF uptake may be clinically useful as it causes less patient discomfort.