Radiosensitization of noradrenaline transporter-expressing tumour cells by proteasome inhibitors and the role of reactive oxygen species

Bortezomib was a gift from Millenium Pharmaceuticals (Cambridge, MA, USA), MG132 was purchased from Sigma-Aldrich (Dorset, UK) and topotecan from Axxora UK Ltd. (Nottingham, UK). All cell culture media and supplements were purchased from Life Technologies Ltd. (Paisley, UK), and all other chemicals were from Sigma-Aldrich (Dorset, UK). No-carrier-added ¹³¹I-MIBG was prepared using a solid-phase system wherein the precursor of MIBG was attached to an insoluble polymer via the tin-aryl bond [28].

Human neuroblastoma-derived SK-N-BE(2c) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The UVW cell line was derived from a human glioblastoma [29]. Cell lines were authenticated in-house using the AmpF/STR Identifiler kit (Applied Biosytems, Warrington, UK). SK-N-BE(2c) cells were maintained in DMEM containing 15% (v/v) fetal calf serum (FCS). UVW cells were transfected to express the NAT gene, facilitating the active uptake of MIBG, as previously described [30] and were maintained in MEM, containing 10% (v/v) FCS and 1 mg/ml geneticin. Transfectants were designated as UWV/NAT.

Cells were seeded in 25-cm² flasks at 10⁵ cells/flask. When cultures were in exponential growth phase, medium was removed and replaced with fresh medium containing the proteasome inhibitors bortezomib or MG132, the antioxidants N-acetyl-L-cysteine (NAC, 1 mM) or tiron (4,5-Dihydroxy-1,3-benzenedisulfonic acid disodium salt monohydrate, 1 mM), or various combinations of these agents. This enabled a comparison of NAC and tiron with respect to the contribution of ROS generation to the cytotoxicity of the proteasome inhibitors. Cells were incubated with drugs for 24 h at 37 C in 5% CO₂. In separate treatments, cells were exposed to X-rays using an RS225 irradiator (Xstrahl, Surrey, UK) at a dose-rate of 1.33 Gy/min, then incubated for 24 h at 37°C in 5% CO₂. After treatment, cells were seeded for clonogenic assay as previously described [3, 4].

The cytotoxic interaction between bortezomib and radiation was examined using clonogenic assay and combination index analysis, according to the method of Chou and Talalay [31]. In this analysis, the toxicity induced by single drugs and scheduled combinations is investigated using the equation CI = (D)₁/(Dx)₁ + (D)₂/(Dx)₂, where (D)₁ and (D)₂ are the doses of each agent which inhibit x% of cell growth when used in combination and (Dx)₁ and (Dx)₂ are the doses of each drug which inhibit x% of colonies when used as single agents.

Initially, exponentially growing cells were treated with each agent alone to determine effective doses. Cells were subsequently treated with a range of doses of bortezomib and radiation, using a fixed dose ratio of bortezomib to radiation, so that the proportional contribution of each agent in the mixtures would be the same at all treatment intensities. The fixed dose ratio was equivalent to 7.6 nM bortezomib:3.8 Gy X-radiation, based on their respective IC₅₀ values. For combinations of ¹³¹I-MIBG and bortezomib, the fixed dose ratio was 7.6 nM bortezomib:1.5 MBq ¹³¹I-MIBG. For the purposes of combination index analysis, simultaneous treatment with {¹³¹I-MIBG + topotecan} was considered as one agent, and the fixed dose ratio was 7.6 nM:0.5 MBq:4.9 nM (bortezomib:¹³¹I-MIBG:topotecan), as this dose killed 50% of clonogens when administered simultaneously in combination. Three different treatment schedules were assessed: bortezomib given 24 h before, after or simultaneously with radiation. The effectiveness of combinations of bortezomib and radiation was quantified by determining a combination index (CI) at various levels of cytotoxicity. CI < 1, CI = 1 and CI >1 indicate synergism, additivity and antagonism, respectively.

Six-week-old female, congenitally athymic mice of strain CD1 nu/nu were obtained from Charles River plc (Kent, UK). In vivo experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986. Tumours in athymic mice formed from SK-N-BE(2c) and UVW/NAT cells express the NAT enabling active uptake of ¹³¹I-MIBG. Subcutaneous tumour growth was established as previously described [3]. Mice were used for experimental therapy when the tumour volumes had reached approximately 100 mm³. To monitor potential toxicity, experimental animals were examined daily for signs of distress and weighed weekly. Mice were randomized into treatment groups, each consisting of six animals that received: PBS solution; 0.8 mg/kg bortezomib solution; simultaneous administration of ¹³¹I-MIBG (18 or 5 MBq for SK-N-BE(2c) or UVW/NAT, respectively) and topotecan (1.75 or 0.875 mg/kg for SK-N-BE(2c) or UVW/NAT, respectively); or administration of bortezomib 24 h after {¹³¹I-MIBG + topotecan} - all by i.p injection. The indicated activities of ¹³¹I-MIBG administered to the mice were shown previously by us to induce significant delay of growth but incomplete sterilization of SK-N-BE(2c) and UVW/NAT xenografts, and the simultaneous administration of ¹³¹I-MIBG + topotecan was demonstrated to be the most effective schedule [3]. Bortezomib doses (0.5 to 1 mg/kg) were similar to those used in previous preclinical studies [9]. Tumours were measured with callipers immediately before treatment and twice weekly thereafter. On the assumption of ellipsoidal geometry, diameter measurements were converted to an approximate tumour volume by multiplying half the longest diameter by the square of the mean of the two shorter diameters.

Data are presented as means ± standard error of the mean (SEM), unless otherwise stated, with the number of independent repetitions provided in the legend to each figure. Statistical significance was determined using Student's t test. A P value < 0.05 was considered to be statistically significant and < 0.01 highly significant.

When given as a single agent at a dose of 1 to 30 nM, bortezomib decreased the survival of clonogens of both SK-N-BE(2c) cells and UVW/NAT cells (Figure 1A) in a concentration-dependent manner. Following treatment of either cell line with bortezomib at concentrations ≥ 10 nM, clonogenic cell kill was highly significantly greater than that of untreated control cultures. The decreased clonogenic capacity of SK-N-BE(2c) and UVW/NAT cells resulting from X-irradiation was enhanced by the treatment with bortezomib at 3 and 5 nM (Figures 1B,C). The IC₅₀ values obtained for SK-N-BE(2c) cells exposed to X-radiation alone, or in the presence of 3 or 5 nM bortezomib were 3.72 ± 0.16, 2.82 ± 0.20 and 2.42 ± 0.15 Gy, respectively. For UVW/NAT cells, the IC₅₀ values were 4.23 ± 0.02, 2.94 ± 0.12 and 2.73 ± 0.11 Gy for X-radiation alone and 3 and 5 nM bortezomib, respectively. These results indicate dose enhancement ratios at the 50% level of toxicity (DER₅₀), in SK-N-BE(2c) cells and UVW/NAT cells respectively, of 1.44 and 1.32 for 3 nM bortezomib, and 1.54 and 1.55 for 5 nM bortezomib.

Representative CI values for treatment of SK-N-BE(2c) and UVW/NAT cells with bortezomib and X-radiation are shown in Table 1. These indicated that supra-additive clonogenic cell kill (CI < 1) was dependent on both the dose and the schedule. Synergism was most appreciable following treatment with high dosage of both agents and the administration of bortezomib 24 h after X-radiation (Table 1).

Similarly, the interaction between bortezomib and ¹³¹I-MIBG suggested that the administration schedule was an important determinant of synergism. For SK-N-BE(2c) and UVW/NAT cells, synergism was evident in response to the administration of bortezomib 24 h after ¹³¹I-MIBG or simultaneous treatment with both agents (Table 2). Administration of bortezomib 24 h before ¹³¹I-MIBG had an antagonistic effect (CI > 1) upon the toxicity to both cell lines at all combination doses.

Three-way combination treatment consisted of bortezomib and {¹³¹I-MIBG + topotecan} - the latter two agents being given simultaneously [3]. Supra-additive clonogenic cell kill was observed only when bortezomib was administered 24 h after {¹³¹I-MIBG + topotecan} (Table 3).

In vitro experimental results indicated no enhancement of tumour cell kill by scheduling bortezomib 24 h before {¹³¹I- MIBG + topotecan} or simultaneous administration of the components of the triple combination. Therefore, in order to reduce the number of experimental animals, the latter schedules were not administered to athymic mice bearing xenografts. None of the animals in this study showed sign of distress. The effects of agents administered alone or in combination on the growth in athymic mice of xenografts derived from SK-N-BE(2c) and UVW/NAT cells are shown in Figure 2. Bortezomib alone induced a slight delay in the growth of SK-N-BE(2c) tumours. The time taken to increase tumour volume fivefold (τ ₅) was 14.7 days, compared to untreated control τ ₅ of 12.3 days. Simultaneous administration of {¹³¹I-MIBG + topotecan} induced a similar delay in the growth of SK-N-BE(2c) xenografts (τ ₅ = 15.8 days). However, bortezomib administered 24 h after {¹³¹I-MIBG + topotecan} resulted in enhanced tumour growth delay, manifest by a τ ₅ value of 28.5 days.

In xenografts derived from UVW/NAT cells, bortezomib alone had no effect on growth rate, exemplified by τ ₅ values of 16.0 and 15.9 days for untreated control groups and bortezomib, respectively. In contrast, simultaneous administration of ¹³¹I-MIBG and topotecan induced an enhancement of growth delay (τ ₅ = 18.2 days) compared with PBS-treated controls. Bortezomib administered 24 h after {¹³¹I- MIBG + topotecan} resulted in a failure by tumours to achieve a fivefold increase in volume over 42 days. Therefore, in xenografts derived from either SK-N-BE(2c) or UVW/NAT cells, the triple combination, consisting of bortezomib administered 24 h after {¹³¹I- MIBG + topotecan}, induced significantly greater growth delay than bortezomib alone or the ¹³¹I-MIBG + topotecan combination.

The mechanism of bortezomib-induced clonogenic cell kill was investigated by determining the protection afforded by treatment with antioxidants. The results are shown in Figure 3. In both SK-N-BE(2c) and UVW/NAT cells, the magnitude of bortezomib-induced clonogenic cell kill was diminished by NAC. For example, exposure to 10 nM bortezomib reduced clonogenic survival to 20% (SK-N-BE(2c) cells) or 49% (UVW/NAT cells) of control values, whereas in the presence of 1 mM NAC, the corresponding values were 83% and 93%, respectively. This suggests that a significant proportion of the cytotoxicity induced by bortezomib as a single agent was due to ROS. An alternative ROS scavenger, tiron, completely blocked bortezomib-induced cytotoxicity at all concentrations (Figure 3), suggesting a different mechanism of action of NAC and tiron or a different degree of nullification of ROS.

MG132, an alternative inhibitor of proteasome activity, may have a pharmacologic profile different from that of bortezomib. Single agent treatment with MG132 caused a concentration-dependent reduction in the survival of SK-N-BE(2c) and UVW/NAT clonogens (Figure 4A). MG132 also sensitized both cell lines to radiation treatment (Figure 4B,C). The IC₅₀ values obtained for SK-N-BE(2c) cells following X-irradiation alone or with simultaneous administration of 150 or 200 nM MG132 were 3.72 ± 0.17, 2.70 ± 0.18 and 2.15 ± 0.09 Gy, respectively. For irradiated UVW/NAT cells, the corresponding IC₅₀ values were 4.23 ± 0.16, 2.36 ± 0.09 and 2.00 ± 0.20 Gy. These results indicate dose enhancement ratios at the 50% level of cell kill (DER₅₀), in SK-N-BE(2c) cells and UVW/NAT cells, respectively, of 1.38 and 1.79 for 150 nM MG132, and 1.73 and 2.12 for 200 nM MG132. These DER₅₀ values are comparable to those obtained following treatment with bortezomib, which ranged from 1.32 to 1.55. This suggests that although the two agents had a similar effect, the concentrations required differed by approximately 40-fold. This difference in potency between bortezomib and MG132 has previously been reported [13, 32, 33], though not fully explained.

The dose-dependent kill of SK-N-BE(2c) or UVW/NAT clonogens observed following treatment with MG132 was not significantly altered by simultaneous treatment with the antioxidants NAC or tiron (Figure 5). Therefore, in contrast to the clonogenic cell kill resulting from bortezomib treatment, MG132-induced kill was not mediated by ROS. This suggests that although bortezomib and MG132 both target the proteasome, both induce clonogenic kill as single agents and both sensitize cancer cells to ionizing radiation, their mechanisms of cytotoxicity do not both involve the generation of ROS.