Effects of novel and conventional anti-cancer agents on human endothelial permeability: influence of tumour secreted factors.

A number of anti-cancer agents have been implicated in vascular toxicity. The effects have been attributed to direct drug toxicity towards endothelium. Little attention has been focussed on the interaction between anticancer drugs, endothelial cells and tumour secreted factors. It is well known that tumours can secrete factors such as vascular permeability factor which do affect endothelial cells and could alter their response to the vascular effects of anticancer drugs. In the present study, we have examined, in vitro, the direct effects of vinblastine (VBL), 5-fluorouracil (5-FU), melphalan (L-PAM) and the novel tubulin inhibitor combretastatin A-1 (CBS) on endothelial permeability under normal and tumour simulated conditions. Monolayers of human umbilical vein endothelial cells (HUVEC) grown on membrane filters were incubated in drug in normal growth medium or medium conditioned by the human melanoma cell line, RPMI-7951 (TCM). VBL caused a rapid increase in permeability during the first 20 minutes, which was maintained for the duration of the experiment (120 minutes). The effect was not altered by TCM or restored to control levels when VBL was replaced by drug-free medium. Similarly, CBS caused a rapid increase in permeability; however, in contrast to VBL, this increase was enhanced by TCM. The changes induced by VBL and CBS were accompanied by contraction of the endothelial F-actin cytoskeleton. Neither L-PAM nor 5-FU altered the permeability of HUVEC monolayers. This study demonstrates that certain anti-cancer agents have a direct effect on endothelial cells, leading to an increase in the permeability of endothelial monolayers. Both VBL and CBS have vascular components in their mode of action which may lead to vascular collapse and tumour necrosis.

Changes in coagulation and permeability properties of human endothelial cells in vitro induced by TNF-alpha or 5,6 MeXAA.

5,6 dimethyl xanthenone acetic acid (5,6 MeXAA), an analogue of flavone acetic acid (FAA), has been shown to be more active against murine tumours than FAA. As both drugs have a vascular component in their mechanism of action similar to that observed for TNF-alpha, we have studied the effects of 5,6 MeXAA alone and in combination with TNF-alpha on endothelial function in vitro. The changes induced by the drugs on procoagulant activity and permeability were determined under tumour-simulated conditions of low oxygen tension and the presence of tumour-secreted factors. Procoagulant activity was assayed by measuring the time taken for human umbilical vein endothelial cells (HUVECs) to clot normal human plasma, increased activity resulting in reduced clotting times. HUVECs incubated under aerobic conditions were more sensitive to TNF-alpha than cells incubated at < or = 0.2% oxygen. Culture medium conditioned by the human breast adenocarcinoma cell line MDA-MB-231 strongly upregulated procoagulant activity under both aerobic and hypoxic conditions; clotting times were further reduced by TNF-alpha. Both 5,6 MeXAA and FAA potentiated the effect of TNF-alpha on normal hypoxic endothelial cells; however, under all other conditions, neither drug in combination with TNF-alpha upregulated clotting activity. The presence of tumour-secreted factors had a far greater effect on upregulating procoagulant activity than did oxygen tension. In contrast to procoagulant activity, permeability was insensitive to TNF-alpha and low concentrations of 5,6 MeXAA also caused no change in permeability.

Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue.

A method for the isolation and long-term culture of human microvessel endothelial cells from mammary adipose tissue (HuMMEC) obtained at breast reduction surgery has been developed. Pure cultures of HuMMEC were isolated by sequential digestion of the fat with collagenase and trypsin followed by specific selection of microvessel fragments with Ulex europaeus agglutinin-1 coated magnetic beads (Dynabeads). The resulting cells formed contact-inhibited monolayers on gelatin and fibronectin substrates and capillary-like "tubes" on Matrigel; they also expressed von Willebrand factor, angiotensin-converting enzyme, and accumulated acetylated low density lipoprotein. Further immunofluorescence characterization revealed the presence of antigens for the endothelial cell specific monoclonal antibodies EN4 and H4-7/33. In addition, the origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (CD31), and endothelial leukocyte adhesion molecule-1 (ELAM-1/E-selectin) upon stimulation with tumor necrosis factor (TNF) alpha. HuMMEC were found to express-1 ELAM-1 at lower levels of TNF alpha (< 10 ng/ml) than required by human umbilical vein endothelial cells. These cells should provide a useful in vitro model for studying various aspects of microvascular biology and pathology.

Flavone acetic acid induced changes in human endothelial permeability: potentiation by tumour-conditioned medium.

Flavone acetic acid (FAA) causes significant regression of larger established tumours in murine in vivo systems. This in vivo effect of FAA has been shown to include a vascular component. In an effort to elucidate the mechanism of action of FAA, we have studied the effects of FAA on the permeability of human endothelium in vitro. Monolayers of human umbilical vein endothelial cells (HUVEC) grown on polycarbonate filters were incubated in 1 mg/ml FAA for 120 min at 37 degrees C. During the first 60 min, there was a 6-8-fold increase in permeability; this was followed by a return to control levels even in the continued presence of FAA. In contrast, in the presence of tumour conditioned medium, FAA caused a rapid 6-fold increase in permeability which did not subsequently return to control levels. The permeability changes which occurred under the latter conditions were accompanied by a rapid contraction of the cytoskeleton. The permeability of monolayers of human melanoma cells was unaffected by FAA.

Flavone acetic acid as a modifier of endothelial cell function.

Flavone acetic acid (FAA) causes significant regression of larger established tumors in murine systems in vivo, but is only slightly toxic in vitro. This in vivo effect is thought to be indirect, or immunological, rather than a direct cytotoxic effect on tumor cells. Using the WHFIB fibrosarcoma, which grows both in vivo and in vitro, and the murine endothelial cell line B10, we have studied the effect of FAA on the survival of tumor and endothelial cells in vitro. The times taken for 1 mg ml-1 FAA to reduce survival to 0.1 surviving fraction were 63 hr for B10 and greater than 85 hr for WHFIB in vitro. WHFIB tumors in vivo were more sensitive than tumor cells in vitro, a single dose of 150 mg kg-1 FAA inducing a tumor growth delay of 10 days at treatment size + 2 mm. As FAA is more toxic to tumor-bearing animals than to those which are non-tumor bearing the effect of tumor conditioned medium on the cytotoxicity of FAA toward B10 cells was studied; no enhanced effect was seen. As FAA is only weakly cytotoxic in vitro to endothelial cells, and even less so to tumor cells, sublethal effects of FAA on endothelial cell function in vitro were studied. The permeability of monolayers of human unbilical vein endothelial cells (HUVEC) in vitro is transiently increased by FAA. Also, procoagulant activity of HUVEC is induced by FAA and this activity is further enhanced in the presence of a factor isolated from Meth-A tumor cells.

Radiosensitization by misonidazole, pimonidazole and azomycin and intracellular uptake in human tumour cell lines.

The radiosensitization of two human tumour in vitro cell lines, HT-1080 and LoVo, has been compared with that of the Chinese hamster cell line V79-379A. Although the two human tumour cell lines were more radiosensitive than the V79 cell line sensitizer, enhancement ratios for misonidazole, pimonidazole and azomycin were similar (relative to extracellular concentration) for all three cell lines. Average intracellular concentrations of radiosensitizer were measured by high-performance liquid chromatography. In all three cell lines the uptake of misonidazole and azomycin was extremely rapid whereas that of pimonidazole was initially much slower before reaching a plateau. The ratios of intracellular concentration of radiosensitizer to extracellular concentration (Ci to Ce) for misonidazole were 0.8 (HT-1080) and 0.7 (LoVo and V79); for azomycin 0.9 (HT-1080 and LoVo) and 0.8 (V79). In contrast Ci/Ce for pimonidazole varied with cell line, the values being 1.8 (LoVo), 2.6 (HT-1080) and 3.3 (V79). Intracellular amounts of non-protein sulphydryl (NPSH) varied between cell lines by about a factor of three. However, when the average cell volume was taken into consideration the concentrations of NPSH were very similar, being 4.2 (HT-1080), 5.6 (LoVo) and 5.7 (V79) mmol dm-3. NPSH levels expressed as nmol per mg protein were also similar.

Radiosensitizer-DNA interactions in relation to intracellular uptake.

We have studied the intracellular uptake of a number of neutral, acidic, and basic radiosensitizers. For neutral sensitizers, we observed a correlation between the measured intracellular concentration and sensitization, but for bases, a large change in average intracellular concentration results in only a small change in sensitization. In addition, by modifying the intralysosomal pH, we have altered the measured average intracellular concentration of the weak base pimonidazole by a factor of two, although this had no detectable effect upon sensitization. Using spin filtration of solutions of sensitizers with naked calf thymus DNA or chromatin we have assessed the affinity of DNA for sensitizers with different prototropic and lipophilic properties. We have also shown that this anomalous behavior of the basic sensitizers could be partly explained on the basis of intracellular localization adjacent to the DNA due to ionic interactions. Thus, intracellular localization needs to be considered when interpreting average intracellular uptake data.

A comparison of colorimetric and clonogenic assays for hypoxic-specific toxins with hamster and human cells.

The hypoxic cytotoxicities of misonidazole and pimonidazole (Ro 03-8799) towards the human tumor cell lines HT-1080 and LoVo have been compared with those seen with Chinese hamster V79-379A cells. Survival was assayed using two colorimetric assays, either a tetrazolium salt (MTT) or methylene blue, and by conventional colony scoring. The drugs were more cytotoxic towards HT-1080 and LoVo cells than V79 cells. The times taken for 10 mmol dm-3 misonidazole to reduce survival to 0.1 surviving fraction (SF) using colony formation as the end point were 2.6 hr for HT-1080, 2.4 hr for LoVo, and 3.5 hr for V79; using the MTT assay these times were 3.5 hr, 2.1 hr, and 2.9 hr, respectively. The times for 2 mmol dm-3 pimonidazole to reduce survival to 0.1 SF using colony formation as the end point were 2.0 hr for HT-1080, 1.7 hr for LoVo, and 3.7 hr for V79; using the MTT assay these times were 2.5 hr, 1.4 hr, and 2.5 hr, respectively.

Rapid-mixing studies of radiosensitivity with thiol-depleted mammalian cells.

A moderate reduction in the non-protein thiol content of V79 379A Chinese hamster cells, obtained by pretreatment with buthionine sulphoximine (BSO), diethyl maleate (DEM) or N-ethyl maleimide (NEM), increase both the absolute radiosensitivity of the cells in hypoxia and the radiosensitizing effect of adding oxygen 7 ms after irradiation. Combined pretreatment of cells with BSO and NEM removes most of the non-protein thiol and some of the protein thiol; such treatment further increases the radiosensitivity of hypoxic cells but there is no further effect of adding oxygen 7 ms after irradiation. Addition of 2-mercaptoethanol to cells 7 ms after irradiation gives protection factors that increase with increasing severity of thiol depletion. Substantial radioprotection can still be observed when 2-mercaptoethanol is added 70 ms after irradiation of cells pretreated with BSO and NEM; there is no effect of adding 2-mercaptoethanol to such cells 50s after irradiation. These observations support the repair-fixation model of radiation damage and suggest that, in addition to the established role of non-protein thiol in chemical repair of radiation damage, other endogenous reducing agents such as protein thiol may be important in determining cellular radiosensitivity. A relatively long-lived thiol-modifiable component of radiation damage has been observed within hypoxic thiol-depleted cells.

A comparison of the intracellular uptake and radiosensitization efficiency in different media of uncharged 2-nitroimidazoles of varying lipophilicity.

The effect of varying octanol: water partition coefficients, P, (range 0.026-260) on the uptake of uncharged 2-nitroimidazoles into Chinese hamster V79 379A cells has been studied. Average intracellular concentrations were measured by high performance liquid chromatography after centrifuging cells through oil or an aqueous medium. The ratio of intracellular concentration of radiosensitizer to extracellular concentration (Ci/Ce) for misonidazole (P = 0.43) was 0.85 for the oil method and 0.68 for the aqueous method. For values of P less than about 0.05 uptake was initially very slow and Ci was always less than Ce. When P greater than or equal to 0.1 uptake was rapid and then remained unchanged for times up to 3 h; for P greater than or equal to 10, Ci/Ce increased rapidly as P increased. Ro 31-1405 (P = 260) concentrated by a factor of 7 inside the cell. Although uptake was identical for cells suspended in full growth medium and PBS, radiosensitization was greater for cells in PBS: 1 mmol dm-3 misonidazole produced an enhancement ratio of 1.6 in full growth medium and 1.9 in PBS. This increase in radiosensitization could not be accounted for by protein binding. However, measurements on cellular non-protein sulphydryl (NPSH) demonstrated the levels to be reduced to about 60 per cent for cells in PBS. Similar reductions in NPSH levels have previously been shown not to increase the radiosensitivity of control cells but to increase greatly the effectiveness of nitroimidazole radiosensitizers.

Radiosensitization of Chinese hamster cells by oxygen and misonidazole at low X-ray doses.

The radiosensitization of Chinese hamster V79 cells in vitro by air and misonidazole at low X-ray doses (0.2-6.0 Gy) had been studied. These survival data, together with high-dose data, were fitted to the linear quadratic model ln S = -(alpha D + beta D2), deriving estimates of alpha and beta by six different methods to illustrate the influence of the statistical treatment on the values so derived. This in vitro study clearly demonstrated that the survival parameters alpha and beta are dependent to some degree on the method of analysis of the raw survival data; however, their ratios, the values of oxygen enhancement ratios (OERs) and radiosensitizer enhancement ratios (SERs) derived from the different methods, are similar. All methods of analysis give reduced OERs at low radiation doses for combined low- and high-dose X-ray data. However, the OERs are still appreciably high, ranging from 2.45 to 2.50 for an oxic dose of 2 Gy. All methods of analysis gave reduced SERs at low doses for combined low and high X-ray dose data for hypoxic cells irradiated in 1 mmol dm-3 misonidazole. At survival levels corresponding to doses of 2 Gy in the presence of 1 mmol dm-3 misonidazole and SERs ranged from 1.2 to 1.5.

Glutathione-reactive nitro compounds as radiosensitizers: mechanistic and therapeutic implications.

The "extra" radiosensitization seen with GSH-reactive nitro compounds is too large to be accounted for by GSH-depletion acting independently--there must be competition. The GS-conjugate leaks out of cells slowly and is trapped at high concentrations. Its properties, such as concentration trapped and reduction potential, must be considered. Limited therapeutic exploitation of the glutathione conjugate trapping and concomitant GSH depletion may be possible if intratumor injection is permitted.

Cellular uptake of misonidazole and analogues with acidic or basic functions.

Average intracellular concentrations of five radiosensitizers in hamster fibroblast-like V79-379A cells in vitro were measured by high performance liquid chromatography, varying the extracellular pH (pHe) and estimating the apparent intracellular pH from the distribution of 5,5-dimethyloxazolidine-2,4-dione. The intracellular: extracellular concentration ratio for the 2-nitroimidazole, misonidazole was constant at about 0.7 for pHe = 6.6-7.6, whereas the weak base, Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol) was concentrated intracellularly at pHe = 7.3-7.4 by a factor of 3.3, the factor increasing from about 0.8 at pHe = 6.0, to 7.5 at pHe = 7.85. The weak acid, azomycin (2-nitroimidazole) showed approximately constant uptake (factor 1.1) between pHe = 6.0-7.0, decreasing to 0.8 at pHe = 7.3 and 0.4 at pHe = 7.8. Measurements of intracellular uptake of Ro 31-0052 (the more hydrophilic and less basic 3'-hydroxypiperidino analogue of Ro 03-8799) and of Ro 31-0258 (3-(2-nitro-1-imidazolyl)propionic acid, a stronger acid than azomycin) were made for comparison. The results were compared with theoretical calculations of pH-induced concentration gradients; the time dependence of the uptake of the bases is not at present clearly understood. These measurements of uptake are broadly consistent with the distribution of misonidazole and Ro 03-8799 in human and animal tissues and provide a useful insight into the likely intracellular concentrations in the clinical use of Ro 03-8799 or other basic radiosensitizers. The measurements also resolve the apparent discrepancy in radiosensitizer efficiency for weak bases in vitro and in vivo which has been previously noted.

The effect of extracellular pH on radiosensitization by misonidazole and acidic or basic analogues.

The effect of extracellular pH (pHe) on the radiosensitization of hypoxic Chinese hamster V79 cells in vitro by the 2-nitroimidazole, misonidazole, and analogues substituted with basic or acid functions has been studied. Misonidazole (1 mmol dm-3) gave an enhancement ratio (e.r.) of 1.6 which remained unchanged over the pHe range of 3.8-9.5. Control hypoxic survival curves in the absence of sensitizer also remained essentially unchanged over this pHe range. These results contrast with those seen for 0.1 mmol dm-3 Ro 03-8799 (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanol), a base with pKa = 8.9): the ER increased from 1.4 to 2.1 as pHe increased from 5.6 to 8.4. However, with the weaker bases, Ro 03-8800 and nimorazole (morpholino derivatives with pKa = 6.3 and 5.2 respectively) the e.r. remained constant over a wide pHe range. Nitroimidazoles substituted with acidic functions gave decreasing sensitization with increasing pHe. For azomycin (pKa = 7.2) at 1 mmol dm-3 the e.r. decreased from 1.9 at pHe 4 to 1.0 at pHe 9. The effect of the proton conductor carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 10 mumol dm-3) on radiosensitization by Ro 03-8799 (0.1 mmol dm-3) and misonidazole (1.0 mmol dm-3) was also studied. At pHe 6.67 the e.r. for Ro 03-8799 was increased from 1.36 to 1.76 by the presence of CCCP, whereas at pHe 7.33 the e.r. was unchanged. In contrast the e.r. for misonidazole was unchanged at pHe 6.65 and 7.33. These results are consistent with pH differentials across the cell membrane creating intracellular:extracellular concentrations gradients for radiosensitizers with acidic or basic functions.

Synthesis and activity of novel nitropyrazines for use as hypoxic cell radiosensitizers.

A series of eight novel nitropyrazines has been prepared by oxidation of sulfoximine intermediates. The partition coefficient, one-electron reduction potential, sensitizer enhancement ratio, and chronic and acute aerobic cytotoxicity have been measured for each. Two representatives of this series were tested in the Ames test and were not found to be mutagenic.

Glutathione depletion enhances the lifetime of oxygen-reactive radicals in mammalian cells.

When the cellular glutathione content is reduced, adding oxygen (130 mumol dm-3) 7 ms after irradiation of hypoxic cells increases the radiosensitivity (factor approximately 1.25), whereas it has much less effect in normal cells.