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Tumor Biology |
Laboratory of Medicinal Chemistry and Radiopharmacy [B. F. J., B. G.], Laboratory of Biomedical Magnetic Resonance [B. F. J., R. J. D., B. G.], Radiobiology and Radioprotection Unit [V. G.], Laboratory of Pharmacotherapy [P. S., O. F.], and Laboratory of Pharmacokinetics, Metabolism, Nutrition and Toxicology [V. P. V., N. D.], Université Catholique de Louvain, B-1200 Brussels, Belgium, and EPR Research Center for the Study of Viable Biological Systems, Dartmouth Medical School, Hanover, New Hampshire 03755 [J. O.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We provide evidence that insulin increases the local pressure of oxygen of tumors (from 0–3 mm Hg to 8–11 mm Hg) as well as the tumor response to irradiation (increasing regrowth delay by a factor of 2.11). We found that the insulin-induced increase of tumor pressure of oxygen: (a) is not caused by an increase in the tumor blood flow, which is even decreased after insulin infusion; (b) is because of a decrease in the tumor cell oxygen consumption (in vivo insulin consumed oxygen three times slower than control cells); and (c) is inhibited by a nitric oxide (NO) synthase inhibitor, N
-nitro-L-arginine methyl ester, when injected i.p. at 15 µmol/kg-1, 1 h before insulin infusion. We demonstrate by immunoblotting that the NO pathway involves a phosphorylation of endothelial NO synthase and showed a concomitant increase in the cyclic GMP tumor level.
These findings provide unique insights into biological processes in tumors, new possible management for treating cancer patients, and raise major questions about the role of insulin secretion (fasting status and diabetes) in the clinical response of tumors to radiation therapy.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initially, we made the assumption that insulin could be an important modulator of tumor oxygenation, because this hormone is known to change blood flow (oxygen supply; Refs. 7, 8, 9 ). In recent years, several studies have shown that hyperinsulinemia can increase blood flow in human skeletal muscle in vivo (10) . They reported that i.v. insulin caused a dose-dependent increase in the rate of resting leg blood flow in humans, independent of hypoglycemia (11) . A positron emission tomography study with 15O water showed that hyperinsulinemia almost doubled skeletal muscle blood flow (12) . Because the tumor models we used were implanted in the skeletal gastrocnemius muscle, we wanted to know if the tumor oxygenation could increase because of an increase in tumor blood flow, as it did in the muscle. Using two different tumor models, we demonstrate that insulin has a profound effect on tumor oxygenation. We actually found that the increase in tumor oxygenation was not because of an increase in tumor blood flow but because of a decrease in tumor cell oxygen consumption. We also demonstrated that the increase in tumor oxygenation was NO-dependent, because the effect was inhibited by the NOS inhibitor L-NAME. We also identified that the NO pathway involves a phosphorylation of eNOS and showed a concomitant increase in the cGMP tumor level. Finally, we found that insulin infusion increases the sensitivity of tumors to irradiation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment
Anesthesia was first induced by an i.p. injection of ketamine (80 mg/kg)/xylazine (8 mg/kg) and maintained with ketamine alone (30 mg/kg). Insulin (Actrapid HM; Novo Nordisk, Bagsvaerd, Denmark) was infused i.v. at a rate of 16 mU/kg/min for 25 min. The dose of insulin was chosen as one known to modify the skeletal muscle blood flow on rodents. Carbogen (5% CO2/95% O2) breathing (5 liter/min) was used as a reference treatment. The control group was infused with a 0.9% NaCl solution only. When used, the NOS inhibitor, N-nitro-L-arginine methyl ester (Sigma, Steinheim, Germany) was injected i.p. at 15 µmol/kg-1, 1 h before insulin infusion (15)
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pO2 and Blood Flow Measurements
Local tumor oxygenation measurements were carried out using two independent techniques: EPR oximetry and a fiber-optic device, OxyLite. We also used two complementary techniques to assess the blood flow inside the tumor: the OxyFlo system and contrast-enhanced MRI. Mice were maintained at 37°C using an homeothermic blanket.
EPR Oximetry.
EPR spectra were recorded using an EPR spectrometer (Magnettech, Berlin, Germany) with a low frequency microwave bridge operating at 1.1 GHz and extended loop resonator. Charcoal (Charcoal wood powder, CX0670–1; EM Science, Gibbstown, NJ) was used as the oxygen-sensitive probe in all of the experiments. Calibrations curves were made by measuring the EPR line width as a function of the pO2. For this purpose, the charcoal was suspended in a tumor homogenate, and EPR spectra were obtained on a Bruker EMX EPR spectrometer (9 GHz) between 0 and 21% O2. Nitrogen and air were mixed in an Aalborg gas mixer (Monsey, NY), and the oxygen content was analyzed using a servomex oxygen analyzer OA540 (16)
. Mice were injected in the center of the tumor (8-mm diameter) using the suspension of charcoal (100 mg/ml, 50-µl injected, 1–25-µm particle size). The EPR measurements were started 2 days after the injection. The tumor under study was placed in the center of the extended loop resonator, which sensitive volume extended 1 cm into the tumor mass, using a protocol described previously (3
, 17)
. The localized EPR measurements correspond to an average of pO2 values in a volume of 
10 mm3 (3)
.
OxyLite/OxyFlo Technique.
We used the OxyLite in conjunction with OxyFlo (Oxford Optronix, Oxford, United Kingdom) for simultaneously and continuously monitoring tissue blood flow, oxygenation, and temperature at the same location (18
, 19)
. Fiber-optic microprobes combining a laser Doppler system, an oxygen-sensor, and a thermocouple were inserted both into the tumor and in the muscle. Data were collected continuously at a sampling frequency of 20 Hz, before, during, and 35 min after insulin infusion. OxyLite pO2 measurements are single point measurements, and the volume sampled is confined to the sensor tip (230-µm diameter). Braun et al. (20)
estimated that the probe would measure an average pO2 in an area of 40,000 µm2.
MR Experiments.
MRI acquisitions were performed with a 4.7 Tesla Bruker Biospec experimental imager. Mice were maintained at 37°C during the experiments by flushing warm air inside the magnet. A preliminary anatomical rapid T2 image [Rapid Acquisition with Relaxation Enhancement (RARE) sequence: time of repetition (TR) = 1500 ms, time of echo (TE) = 11.5 ms, 4 averages, echo train length = 8] was acquired to define a region of interest encompassing the whole tumor. Dynamic Gd-DTPA (FLASH imaging) before and after i.v. administration of insulin were performed: TR = 5 ms, TE = 5 ms, 
; = 5°, matrix size = 64*64, field of view = 6 cm. A Gd-DTPA uptake experiment consisted of 40 consecutive T1-weighted FLASH images with a total acquisition time of 40 s. Gd-DTPA in 0.9% NaCl solution was injected as an i.v. bolus injection (180 µl, 1 s duration, 0.2 mmol/kg) after five precontrast images. Each mouse received a first bolus of Gd-DTPA alone (constituting the control part of the experiment). Signal intensity had decreased to the basal level after 2 h, so the second bolus injection was performed 2 h after the first one. For the treated group, the insulin infusion (25 min) was completed 30 min before the second contrast agent injection. The two uptake curves were then compared (5)
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Oxygen Consumption Rate Evaluation
The method developed by James et al. (21)
was used. All of the spectra were recorded on a Bruker EMX EPR spectrometer operating at 9 GHz. Mice were first treated with insulin in vivo. Thirty min after the end of insulin infusion, tumors were excised, trypsinized for 30 min, and cell viability determined as reported previously (21)
. Cells (2 x 107/ml) were suspended in 10% dextran in complete medium. A neutral nitroxide, 15N 4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl at 0.2 mM (CDN Isotopes, Pointe-Claire, Quebec, Canada), was added to 100-µl aliquots of tumor cells that were then drawn into glass capillary tubes. The probe (0.2 mM in 20% dextran in complete medium) was calibrated at various O2 between 100% nitrogen and air so that the line width measurements could be related to O2 at any value. Nitrogen and air were mixed in an Aalborg gas mixer, and the oxygen content was analyzed using a servomex oxygen analyzer OA540. The sealed tubes were placed into quartz ESR tubes, and samples were maintained at 37°C. As the resulting line width reports on O2, oxygen consumption rates were obtained by measuring the O2 in the closed tube over time and finding the slope of the resulting linear plot.
Irradiation and Tumor Regrowth Delay Assay
The tumor-bearing leg was locally irradiated with 16 Gy of 250 kV X-rays (RT 250; Philips Medical Systems). Mice were anesthetized, and the tumor was centered in a 3-cm diameter circular irradiation field. When tumors reached 8.0 ± 0.5 mm in diameter, the mice were randomly assigned to a treatment group and irradiated. After treatment, tumors were measured every day until they reached a diameter of 16 mm, at which time the mice were sacrificed. A linear fit could be obtained between 8 and 16 mm, which allowed us to determine the time to reach a particular size for each mouse. For each tumor, transversal and antero-posterior measurements were obtained. An average tumor diameter was then calculated.
Metabolic Parameters Quantification
The measurements were performed on FSAII tumors and on leg muscles (before treatment and 30 min after insulin infusion).
ATP Content.
Tumor and muscles slices were taken, washed twice in NaCl solution. and sonicated in 1 ml of 2% perchloric acid. The intracellular ATP content was measured on neutralized perchloric acid extracts using ATP Bioluminescence Assay kit CLS II from Boehringer (Brussels, Belgium).
Lactate Content.
Tumor slices were taken, washed twice in NaCl solution, and sonicated in 2 ml of 3.65% perchloric acid. This deproteinization was performed twice. The lactate content was measured on neutralized extracts using the Lactate kit from Sigma Diagnostics (Bornem, Belgium).
Glycogen Content.
Tumor and muscle slices were taken, rapidly washed twice in NaCl solution, sonicated in 1 ml of 1 M KOH, then heated at 100°C for 10 min. After neutralization with acetic acid and centrifugation, the supernatant was incubated in the presence of 
-amylo glucosidase in pH 5 acetate buffer (1 M). The glucose produced was quantified by an enzymatic reaction as described elsewhere (22)
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Blood Glucose Level.
Blood samples were taken and centrifuged at 4°C. The analysis was performed on serum using the Glucose kit from Elitech Diagnostics (Sees, France).
Identification of the NO Pathway
Immunoblotting.
Insulin-treated or control FSAII-bearing mice were sacrificed, and tumors were homogenized in a buffer containing phosphatase and protease inhibitors (23)
. Samples were equally loaded and processed for immunoblotting as described elsewhere (24)
. P-eNOS antibody was from NEB Cell Signaling Technology (Beverly, MA), eNOS antibody from BD Transduction Labs (Lexington, KY).
cGMP Quantification.
Insulin-treated or control FSAII-bearing mice were sacrificed, and tumors were homogenized in cold 6% (w/v) trichloroacetic acid. Samples were then centrifuged at 2000 x g for 15 min at 4°C. The supernatant was washed four times with 5 volumes of water-saturated diethyl ether. The aqueous extract remaining was dried under a stream of nitrogen at 60°C, and the dried extract was dissolved in a 0.05 M sodium acetate buffer pH 5.8 containing 0.02% (w/v) BSA before analysis. The cGMP content was then determined by the cGMP enzyme immunoassay kit from Biotrak (Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany).
Statistical Analysis
Results are presented as means ± SE. Comparisons between groups were analyzed by t test (two-sided) or ANOVA for experiments with more than two subgroups. In this case, the Dunnet PostHoc test was considered. Ps < 0.05 were considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Table 1
). Prolonged experiments carried out on 4 mice indicate that the tumor pO2 reached its maximum value within 25–30 min after the end of the infusion and fell to the basal value within 6–7 h (data not shown). Carbogen breathing, used as a positive control, induced an increase of 23.2 mm Hg and 18.7 mm Hg for TLT and FSAII tumor models, respectively (n = 10). A typical experiment is shown on Fig. 1
. All of the results are summarized in Table 1
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; n = 4/group; P = 0.021). Because the "control" and "insulin curves" were performed on the same mouse, we avoided external perturbations from different tumor size, vascularization, or contrast agent clearance. A pixel by pixel analysis demonstrated that 32–60% of the pixels contributed to the blood flow decrease of the tumor after insulin infusion. The increase in blood flow in the muscle did not lead to an increase in flow inside the tumor in this case: the increase in normal tissue blood flow because of vasodilation (29.2 ± 6.8% increase in the flow in the muscle after insulin infusion; n = 5; data not shown) shunted away the blood flow from the tumor to the muscle ("steal effect"). Therefore, the increase in pO2 cannot be assigned to an increase of the oxygen supply.
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. The mean slopes were -0.80 µM/min ± 0.02 and -0.26 ± 0.06 µM/min for control and insulin groups, respectively (n = 4 for each group). This means that in vivo insulin pretreated cells consumed oxygen three times slower than control cells. We concluded that the tumor oxygenation increase was because of the decrease of the oxygen consumption by the tumor cells.
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Insulin Increases the Tumor Oxygenation by a NO-dependent Pathway.
We made the hypothesis that the decrease in tumor cell oxygen consumption could be mediated by a NO pathway, because insulin exerts cardiovascular actions that are mediated at least in part by this system. To verify the involvement of NO in the effect of insulin on tumor oxygenation, a group of FSAII tumors was treated with L-NAME 1 h before insulin infusion. We observed that this NOS inhibitor abolished the effect of insulin on tumor pO2 (Fig. 4)
. In addition, we identified by immunoblotting that eNOS was the only detectable NOS isoform in FSAII tumors (not shown) and importantly, was activated by phosphorylation after in vivo insulin infusion. P-eNOS was present in samples obtained from insulin-treated tumors and absent in control ones (n = 3/group; Fig. 5
), whereas there was no significant difference in eNOS abundance between insulin and control groups (P > 0.05). Finally, we showed that the cGMP level of the tumor significantly increased after insulin infusion (P = 0.013) compared with control tumors (Fig. 6)
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). These data indicate that insulin increased the sensitivity of the tumor to X-ray irradiation, increasing regrowth delay by a factor of 2.11 compared with a factor of 1.61 for carbogen, treatment that is currently being used successfully in the clinic (4)
. The L-NAME pretreated group, additionally infused with insulin and irradiated, is similar to the RX group (P > 0.05), with a regrowth delay of 5.1 ± 0.7 days (Fig. 7)
. L-NAME administration before insulin infusion totally inhibited the radiosensitizing effect of insulin. L-NAME administration before irradiation (without insulin treatment) had no effect on the tumor regrowth delay on this tumor model (the "RX" and "L-NAME + RX" curves are similar; Fig. 7
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Corroborating this mechanism, it has been found that eNOS-/- mice were hypertensive but also insulin-resistant, as evidenced by fasting hyperinsulinemia and a 40% lower insulin-stimulated glucose uptake. Insulin resistance in eNOS-/- mice was related specifically to impaired NO synthesis, because in hypertensive mice induced by a model of renovascular hypertension, insulin-stimulated glucose uptake was normal (vascular NO production was evaluated by measurement of plasma concentration of nitrite and nitrate; Ref. 33 ).
Because ketamine has well-described effects on mobilization of glucose reserves and blood glucose levels (34) , the effect of insulin infusion on the tumor pO2 was also tested on mice anesthetized with isoflurane. It was similar to that of ketamine/xylazine anesthetized mice (data not shown).
The action of insulin on vascular endothelium is thought to couple regulation of hemodynamic homeostasis with metabolic homeostasis. Physiologically, most of these vascular effects appear mediated by NO. Our current findings additionally emphasize the critical role of this insulin/NO pathway in tumors. NO has the unique ability to either initiate or arrest tumor formation: this multifaceted role clearly depends on a variety of conditions that exist in the tumor environment. Here, we demonstrate that when acutely controlling its production, e.g., by insulin administration, the properties of NO can be exploited to potentiate the tumoricidal effects of irradiation.
One straightforward application of the present study is to take advantage of the radiosensitizing effect of the slow infusion of insulin. Because insulin clamp (slow insulin infusion) is already used in specific clinical applications for estimation of insulin sensitivity (35 , 36) , the use of such a protocol can be immediately tested in patients. We showed that insulin radiosensitized FSAII tumors in vivo, with a regrowth delay even higher than using the carbogen treatment. We can speculate why insulin is more efficient in radiosensitizing FSAII tumors than carbogen when carbogen induces a greater increase in tumor pO2. Besides the radiosensitizing effect attributable to oxygen, insulin has been shown to inhibit repair of potentially lethal radiation damage. Johnson et al. (37) showed that the average completion time of an excision repair patch varied according to the state of cell culture and that the completion time was extended after treatment with insulin. Subsequently insulin has been shown to inhibit repair of radiation damage and chromosome aberrations (38) . Finally, an additional effect of NO itself cannot be excluded, because NO has been shown to be a radiosensitizer of hypoxic cells in vitro (39) and in vivo (40) .
Drugs that inhibit respiration, such as meta-iodobenzylguanidine were proposed as potential radiosensitizing agents (41) . Mild hyperglycemia, which has also been demonstrated to reduce oxygen consumption (Crabtree effect), has been tested recently in combination with hyperoxic gas (42) . It is reasonable to assume that the increase in pO2 observed after hyperglycemia can at least partly be explained by a stimulation of insulin secretion and a consequent reduction of oxygen consumption. Because insulin acts by decreasing the oxygen consumption of tumor cells, this approach could be complementary to strategies modifying the oxygen supply in the tumor (i.e., carbogen breathing). However, insulin infusion is not indicated as a sensitizing approach for chemotherapy as the perfusion of the tumor was decreased after that treatment.
Besides the effect of acute injection of insulin on the circulation and the oxygenation of the tumor, our study might have implications for additional studies on the role of the physiology of the host on the tumor response to treatments. What could be the role of inadequate secretion of insulin in a chronic situation? Chronic diabetes often is suggested as a complicating factor for damage in normal tissues in the radiation field and even recently claimed as a predictor for late radiation morbidity (43) . Although many factors can be evoked in diabetes mellitus such as alterations in blood viscosity and microvascular occlusive changes, our study isolates one factor with major implications in the tumor biology that should be additionally investigated.
Our results indicate that an important consideration may be the influence of the fasting status of patients before irradiation. Because insulin secretion is dependent on feeding, it is likely that the tumor blood flow and oxygenation will be modified after a meal. Up to now, the influence of fasting was studied only in very few early works on a possible radioprotective effect on whole-body irradiated mice (44 , 45) . The biodistribution of therapeutic or diagnostic radionuclides has been found to be dependent on food ingestion (46) . Again, it is likely that the fasting status and consequent insulin blood concentration should carefully be taken into account in monitoring treatments in animal models as well as in the clinical situation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Belgian National Fund for Scientific Research (FRSM 3.4560.00) and the Fonds Joseph Maisin. B. F. J. and P. S. are Research Assistants of the Belgian National Fund for Scientific Research. O. F. is Research Associate of the Belgian National Fund for Scientific Research. 
2 To whom requests for reprints should be addressed, at Laboratory of Medicinal Chemistry and Radiopharmacy, Avenue Mounier 73.40, B-1200 Brussels, Belgium. E-mail: Gallez{at}cmfa.ucl.ac.be
3 The abbreviations used are: pO2, pressure of oxygen; EPR, electron paramagnetic resonance; MRI, magnetic resonance imaging; cGMP, cyclic GMP; NOS, nitric oxide synthase; L-NAME, N-nitro-L-arginine methyl ester; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; DTPA, diethylenetriaminepentaacetic acid; P-eNOS, phosphorylated endothelial nitric oxide synthase; RX, radiation dose.
Received 11/13/01. Accepted 4/18/02.
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REFERENCES |
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-irradiated mice. Strahlentherapie, 158: 734-738, 1982.[Medline]
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, control group; 
, insulin-treated group. Left, FSA II (n = 5/group); right, TLT (n = 10/group). Bottom, typical tumor pO2 monitored by OxyLite oximetry (before/during/and after insulin infusion). Left, FSA II; right, TLT. Note the significant increase in tumor pO2 for both tumor models; bars, ±SE.
, % of MRI signal enhancement after the second Gd-DTPA bolus injection (n = 4/group). Note the significant decrease in signal enhancement after the second Gd-DTPA injection for the insulin-treated group, whereas both curves are superimposable for the control group; bars, ± SE.
, control group. Mean slopes: -0.80 µM/min ±0.02 and -0.26 ± 0.06 µM/min for control and insulin groups, respectively (n = 4 for each group). In vivo insulin-pretreated cells consumed oxygen three times slower than control cells; bars, ±SE.
), infused with insulin before 16 Gy of RX (
) Each point represents the mean tumor size ± SE of 6 tumors. The regrowth delay to reach 12-mm tumor diameter was 4.8 ± 0.6 days for RX alone, and 9.1 ± 1.1 days for insulin and RX (P = 0.008). Insulin increased the regrowth delay by a factor of 1.9.