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Tumor Microcirculation Group, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom
Abstract
Carbon monoxide (CO), one of the products of heme oxygenase (HO) catalyzed heme degradation, is a vasodilator. The aim of the present study was to clarify the role of HO in blood flow maintenance in tumors. Male BD9 rats bearing subcutaneous transplants of the P22 carcinosarcoma tumor were treated intraperitoneally (ip) with either tin-protoporphyrin IX (SnPP; 45 µmol/kg), a selective inhibitor of HO or copper-protoporphyrin IX (CuPP; 45 µmol/kg), used as a negative control. The extent of HO activity inhibition was measured using a spectrophotometric assay of bilirubin production and blood flow rates to the tumor and a range of normal tissues were assessed using the uptake of the radiolabelled tracer, iodo-antipyrine (125I-IAP). The animals were cannulated under fentanyl citrate/fluanisone (Hypnorm)/midazolam anesthesia. In the P22 tumor, SnPP, but not CuPP, caused a complete inhibition of HO activity 15 min post-treatment. Administration of SnPP 15 min before blood flow measurements reduced tumor blood flow by 17%, with no effects in any of the normal tissues studied. However, CuPP induced a greater reduction in tumor blood flow than SnPP (45% decrease). Furthermore, CuPP caused a reduction in blood flow to the skin and small intestine but a significant increase to skeletal muscle. The current findings conclusively establish only a minor role played by the HO/CO system in the maintenance of blood flow in this tumor system, despite relatively high levels of HO-1 protein and HO activity. The results also highlight the potential usefulness of CuPP as a tumor blood flow modifier.
Key Words: heme oxygenase • carbonmonoxide • cancer • blood flow
The continuous growth and expansion of solid tumors is critically dependent on the establishment of a vascular network providing nutritive blood flow, thereby, making the tumor vasculature an attractive target for cancer therapy (1, 2). Currently, there is still a need to understand the mechanisms that regulate and maintain blood flow to tumors. In their resting state, many tumors are under the influence of a vasodilatory tone and there is substantial evidence that one of the prime mediators of this is nitric oxide (NO)(3). Indeed, in experimental tumor models, inhibition of NO synthase (NOS) has been shown to result in a significant reduction in blood flow (4) and chronic NOS inhibition has been shown to significantly slow tumor growth (5). In the present study, we investigated the role of another vasodilating molecule, CO, whose in vivo production is mainly via HO activity. The role of CO in the regulation of vascular tone was demonstrated in isolated rat tail artery tissues where hemin treatment suppressed phenylephrine-induced vasocontraction, whereas this effect was abolished by either HO inhibition or the CO scavenger, oxyhemoglobin (6). Furthermore, exogenous CO was shown to induce vasodilatation in precontracted isolated vessels from various origins and species. These include rat thoracic aortas (7), canine femoral, carotid and coronary arteries (8), and porcine coronary arteries and veins (9), as well as arterioles isolated from rat gracilis muscle (10) and rat tail arteries (6). Finally, the HO/CO system was also shown to have an important role in the regulation of hepatic vascular tone, whereby administration of the HO inhibitor, zinc–protoporphyrin IX (ZnPP), was shown to suppress CO production and to promote an increase in the perfusion pressure of isolated rat liver (11, 12). Of interest to us is the potential importance of CO in the regulation of vascular tone in tumors and consequently, the possibilities for exploiting this system for modifying tumor blood flow. HO-1, the inducible form of the enzyme, has been reported to be over-expressed in human brain tumors (13), malignant prostate tissue (14), and human gliomas and melanomas (15, 16) as well as in human esophageal squamous cell carcinomas (17). These findings suggest that CO production, resulting from HO activity, could be an important contributor to the maintenance of blood flow in tumors. However, previous studies in our laboratory using the HO inhibitor, ZnPP, have failed to conclusively establish a role for HO in regulating tumor blood flow because of the lack of inhibition of tumor HO activity by ZnPP in vivo (18). The aim of the present study was to further clarify the potential vascular role of endogenous HO in tumors using another competitive HO inhibitor, tin–protoporphyrin IX (SnPP). Initial experiments were conducted to verify the ability of SnPP to inhibit tumor HO activity before determining the effects of the compound on tumor blood flow. Copper–protoporphyrin IX (CuPP) was used as a negative control in these studies since it has been reported to have no inhibitory effects on HO activity in vivo (19) and to be only a poor inhibitor in vitro (20).
Materials and Methods
Tumors and Treatments.
All animal procedures were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and with the approval of the Ethical Review Committee of the Gray Cancer Institute. Early generation transplants of the P22 rat carcinosarcoma were used in this study (21). Tumor pieces were transplanted subcutaneously into the left flank of 8- to 9-week-old male BD9 rats. Treatments were started when the tumor geometrical mean diameter reached 11–14 mm. Tumor-bearing animals were allocated to one of the following treatment groups: SnPP (Affiniti Research Products, UK) at a dose of 45 µmol/kg, CuPP (Affiniti Research Products, UK) at a dose of 45 µmol/kg, or drug vehicle (50 mM sodium carbonate; Sigma, UK). All the compounds were administered intraperitoneally (ip) at 3 ml/kg body weight. Care was taken to shield SnPP and CuPP from light during preparation and injection into the animal. For tissue HO activity measurements, tumors were excised at various time points post-treatment and microsomal fractions were prepared from tissue homogenates by ultracentrifugation.
HO Activity Assay.
HO activity in tumor microsomal fractions was measured using a spectrophotometric assay of bilirubin production. The method has been published previously (22). Briefly, tissue microsomes were added to the following mixture: MgCl2 (2 mM) phosphate-buffered saline (100 mM, pH 7.4; Sigma, UK), rat liver cytosol as a source of biliverdin reductase (3 mg total protein), hemin (10 µM) (Sigma, UK), glucose-6-phosphate (2 mM) (Sigma, UK), glucose-6-phosphate dehydrogenase (0.2 U; Sigma, UK) and NADPH (0.8 mM; Sigma, UK). The reaction was conducted in the dark for 30 min at 37°C and terminated by the addition of chloroform (Sigma, UK). The amount of extracted bilirubin was calculated by the difference in absorption between 464 and 530 nm and an extinction coefficient of 40 mM-1cm-1 was used for bilirubin. The total protein content of the samples was determined using a colorimetric assay according to the manufacturer’s instructions (Bio-Rad, UK) and bovine gamma globulin was used as a standard.
Tissue Blood Flow Rate.
Blood flow rate in tumors and normal tissues was measured 15 min post-treatment using the uptake, over a short infusion time, of the inert and rapidly diffusible radiolabelled tracer iodo-antipyrine (125I-IAP; Institute of Cancer Research, Sutton, UK). This technique has been described previously (21). Briefly, animals were cannulated under Hypnorm/midazolam anesthesia and the tracer was infused via a tail vein catheter over a 30-sec period. During this time, arterial blood samples from heparinized animals were collected at 1-sec intervals into preweighed glass vials via a tail artery catheter. Blood pressure was monitored via the arterial cannula up to the point of blood flow measurement. At 30 sec, the animal was killed by an injection of Euthatal via the second tail vein catheter and the tumor, overlying skin, skin from the contralateral flank, gastrocnemius muscle, spleen, left kidney, small intestine, heart and brain were excised rapidly. Blood and tissue samples were weighed and counted for 125I levels using a Wallac 1282 Compugamma Universal Gamma Counter. Blood flow for each tissue was calculated in ml blood•g-1•min-1 using tissue levels of 125I, arterial levels of 125I derived from arterial blood counts and the blood:tissue partition coefficient for IAP in each tissue. This method is based on the theory devised by Kety for the exchange of inert diffusible tracers between blood and tissues (23).
Statistics.
Significance tests were conducted on the data groups using analysis of variance (ANOVA) followed by a comparison between the specific groups using the Student’s t test. The analyses were conducted using a statistical package (JMP, SAS Institute Inc.). Values of P < 0.05 were considered significant.
Results
In Vivo HO Enzyme Activity Inhibition.
Administration of a single 45 µmol/kg dose of SnPP caused the HO enzyme activity in the P22 tumor to decrease to zero within the first 15 min of treatment. It remained inhibited for at least 1 hr post-treatment (Fig. 1
). However, the same dose of CuPP did not affect the HO enzyme activity at the same time points examined (Fig. 1). This is consistent with previously reported data for this tumor (18).
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, show that a single 45 µmol/kg dose of the selective HO inhibitor, SnPP, tended to reduce blood flow to several tissues, including the tumor, but none of the effects were significant with the numbers of animals used. However, the same dose of CuPP mediated a 45% decrease in tumor blood flow. No significant effects were observed in the examined normal tissues, only in the skin was the effect greater than in the tumor. A smaller but significant reduction in blood flow was obtained in the small intestine. In contrast to any other tissue, blood flow in skeletal muscle was almost doubled in the CuPP-treated group compared to control (Fig. 2). Furthermore, although mean arterial blood pressure and heart rate remained unaffected by SnPP, CuPP increased heart rate to approximately 125% of control. The tendency for CuPP to decrease mean arterial blood pressure did not reach statistical significance (Fig. 3).
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SnPP is a synthetic metalloporphyrin that acts as a competitive substrate for heme in the HO reaction but does not undergo oxidative degradation by the enzyme (24). In vivo, SnPP was shown to inhibit HO in various organs, including liver, spleen, kidney, and skin of the rat (19, 25), although other investigators have reported a lack of inhibition of intestinal HO (26). In this study, SnPP was used to examine the importance of the HO/CO system in the regulation and maintenance of blood flow to the P22 rat carcinosarcoma tumor. We found that SnPP administration at a dose of 45 µmol/kg ip effectively inhibits tumor HO activity in vivo without significant effects on tumor or normal tissue blood flow. Our studies with the HO inhibitor, ZnPP, showed a significant reduction in tumor blood flow by 30% (18). However, under these conditions, tumor HO activity was not inhibited by ZnPP, indicating that the blood flow responses were completely independent from HO activity (18). Indeed, the effects of ZnPP were very similar to CuPP in terms of both lack of effect on HO activity and blood flow response. This is in contrast to the effects of SnPP and CuPP, in the current study, where clear differences were demonstrated, thereby, suggesting distinct mechanisms of action for the two agents. SnPP effectively inhibited HO activity but had no effect on tissue blood flow rate. On the other hand, CuPP, as expected, had no effect on HO activity but significantly reduced blood flow in tumors and in some of the normal tissues examined while increasing blood flow to skeletal muscle. The blood flow effects of CuPP and ZnPP are clearly not the result of HO inhibition and the results for SnPP conclusively establish only a minor role played by the HO/CO system in the maintenance and regulation of blood flow in this tumor model.
The blood flow-modifying effects of CuPP and ZnPP are difficult to interpret but are likely to relate to various reported effects of these agents, which are related to the vasculature and not to HO. For instance, ZnPP (and SnPP) have been shown to inhibit guanylate cyclase (GC) and NOS (27). ZnPP was also shown to abolish the increase in cAMP and cGMP evoked by vasoactive intestinal peptide and atrial natriuretic peptide, thereby, inhibiting relaxation mediated by these agents in rat aorta (28). These studies demonstrate the nonselectivity of these agents in mediating their actions and the difficulty of choosing the most appropriate inhibitor for HO. Furthermore, little is known about the isozyme specificity of these metalloporphyrins. There is some evidence that SnPP is far more inhibitory to HO-2-dependent activity as compared to ZnPP, with both compounds being equally inhibitory to HO-1 activity (29). This could explain the differential effects of SnPP and ZnPP on overall tumor HO activity in case of a differential in HO-1/HO-2 isozyme abundance in this tumor model.
The lack of blood flow effects of SnPP in the P22 tumor was quite surprising in view of the relatively high HO activity in this tumor (18, 30) and the effectiveness of SnPP at inhibiting it (Fig. 1
). We have previously shown that NOS inhibition causes a large reduction in blood flow to the P22 tumor and this pathway may represent the major contributor to blood flow regulation and maintenance in this model (4).
In the present study, SnPP did not mediate any acute changes in tissue blood flow rate. However, this does not exclude the possibility of long-term effects resulting from prolonged HO activity inhibition. Recently, HO-1 has been linked to cell proliferation and angiogenesis whereby over-expression of the HO-1 gene in endothelial cells was shown to stimulate endothelial cell growth and enhance angiogenesis (31). Furthermore, in vivo administration of ZnPP was shown to significantly suppress tumor growth (32).
In conclusion, this study has clearly demonstrated that in the P22 tumor, the HO/CO system plays only a minor vasodilatory role. It is possible to speculate that the HO/CO system could have a more important vascular role in tumors where NO is not the major vasodilatory molecule. The current findings also highlight the important blood flow modifying effects mediated by CuPP. Elucidation of its mechanism of action would allow further understanding of the factors/pathways responsible for the regulation and maintenance of blood flow in tumors.
Acknowledgments
We thank the Gray Cancer Institute staff for care of the animals and Cancer Research UK for funding this work.
Footnotes
1 To whom requests for reprints should be addressed at Tumor Microcirculation Group, Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood Middlesex, HA6 2JR, United Kingdom. E-mail: khelifi{at}gci.ac.uk 
References
-nitro-L-arginine, and the haemoxygenase inhibitor, zinc protoporphyrin IX, on tumour blood flow. Int J Radiat Oncol Biol Phys 42:849–853, 1998.[Medline]
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