Main

All living organisms need to maintain a healthy intracellular redox balance in order to survive and to proliferate.1 Reactive oxygen species (ROS) are natural by-products of aerobic metabolism whose production correlates with normal cell proliferation through the activation of growth-related signaling pathways.2 Exposure to low levels of ROS can stimulate the growth of many types of mammalian cells, whereas scavengers of ROS suppress normal cell proliferation in human and rodent fibroblasts.3, 4 Furthermore, growth factors trigger the production of hydrogen peroxide (H2O2) that leads to mitogen-activated protein kinase activation and DNA synthesis, a phenomenon inhibited by antioxidant molecules.5, 6 Several observations suggest that ROS also participate in carcinogenesis. First, ROS production is increased in cancer cells, and an oxidative stress can induce DNA damages that lead to genomic instability and possibly stimulate cancer progression.7 Second, elevated ROS levels are responsible for the activation of transcription factors, such as NF-κB and AP-1 during tumor progression.8 Several studies have shown that different types of cancer cells such as colon, liver, lung, kidney, prostate and skin cancer cells9, 10, 11 display a high proliferation rate associated with an increased endogenous production of ROS and a downregulation of their antioxidant enzymatic systems.

On the other hand, ROS can also induce the apoptosis/necrosis of cancer cells. Indeed, most anticancer drugs kill their target cells, at least in part, through the generation of high amounts of intracellular ROS that stimulate pro-apoptotic signal molecules.12, 13 Several anticancer agents, such as 5-fluorouracil,14 platinum,9, 12 arsenic trioxide,15 paclitaxel,16 and anthracyclines17 increase the intra-cellular levels of ROS. Tumor cells generate higher levels of ROS compared with normal cells and are therefore more sensitive to the additional oxidative stress caused by anticancer agents.9, 12, 18

Cells also contain a variety of free radical scavenging systems that protect them from the effects of drugs that generate ROS. Catalase and glutathione peroxidase are enzymes that detoxify H2O2. High levels of reduced glutathione, the cofactor of glutathione peroxidase, have been associated with the multidrug resistance phenotype in tumor cells.19 Therefore, compounds that target such protective mechanisms could enhance the activity of anticancer agents and reverse the multidrug resistance phenotype. For example, buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, can increase the cytotoxicity of melphalan by preventing glutathione peroxidase activity.20 Conversely, N-acetylcysteine protects normal cells from the cytotoxic effects of anticancer drugs by preventing the elevation of intracellular H2O2 through its reductive properties.9 SOD enzymes can also affect tumor cell proliferation via their effects on peroxide levels. Thus, the overexpression of SOD in human cancer cell lines or the use of nonpeptidyl mimic of SOD increases H2O2 production and reduces tumor growth.9, 16, 21, 22

Killing cancer cells through the oxidative stress may require compounds that generate ROS and, more appropriately, catalysts that convert the intracellular ROS into a cocktail of cytotoxic chemical species that selectively kills cancer cells.18, 23 Compounds that contain quinones and tellurium are cytotoxic at low concentrations.24 The LAB027 compound that associates a quinone core along with two tellurium atoms can thus be considered as the prototype of pro-oxidative tellurium compounds. However, LAB027 as well as the other multifunctional redox catalysts synthesized so far have always been tested alone and in vitro.

Colon cancer cells are highly sensitive to the oxidative stress18 and are currently treated by 5FU, oxaliplatin and irinotecan, that generate an intracellular stress in cancer cells. The capacity of organotelluride catalysts to generate an intracellular oxidative burst alone and in combination with a pro-oxidative cytotoxic drug, prompted us to test these molecules against colon cancer in vitro and in vivo. Therefore, we investigated the antitumoral activity of the organotelluride catalyst LAB027 alone and in combination with oxaliplatin in vitro and in vivo in an animal model of colon cancer. Because of the therapeutic potential of this compound, an evaluation of its profile of toxicity profile has also been performed.

Results

LAB027 exerts pro-oxidative effects in vitro

Incubation of mouse CT26 and human HT29 colon cancer cell lines with increasing amounts of LAB027, dose-dependently increased the production of O2•− (CT26: P<0.05 with 2 μM LAB027; HT29: P<0.05 with 1 μM LAB027) (Figure 1). A dose-dependent increase in the production of H2O2 was also observed (CT26: P<0.05 with 2 μM LAB027; HT29: P<0.05 with 2 μM LAB027) (Figure 1). Incubation of NIH3T3 fibroblasts with increasing amounts of LAB027 had no effects on the production of O2•− (P=NS), but dose dependently increased the production of H2O2 by 500% (NIH3T3: P<0.05 with 1 μM LAB027) (Figure 1). Incubation of W138 fibroblasts with increasing amounts of LAB027 had no effects on the production of O2•− (P=NS), but dose-dependently increased the production of H2O2 by 260% (W138: P<0.05 with 1 μM LAB027) (Figure 1).

Figure 1
figure 1

Production of hydrogen peroxide (a) and of superoxide anions (b) by CT26, HT29, NIH3T3 and W138 cells incubated with increasing concentrations of LAB027. Results are the mean±S.E.M. of three experiments performed in triplicates

LAB027 alters the activities of antioxidant enzymes in vitro

Incubation of CT26 and HT29 cells with increasing amounts of LAB027 did not significantly alter SOD, catalase and glutathione reductase activities (P=NS compared with untreated cells) (Supplementary Figure 2). By contrast, incubation of NIH3T3 or W138 fibroblasts with increasing amounts of LAB027 dose-dependently increased the production of SOD (P<0.001 for NIH3T3 and P<0.001 for W138 with 4 μM LAB027) and glutathione reductase activities (P<0.001 for NIH3T3 and P<0.001 for W138 with 4 μM LAB027) versus untreated cells (Supplementary Figure 2) but had no effect on catalase levels.

LAB027 exerts cytostatic and cytotoxic effects in vitro

Incubation of CT26 or HT29 cells with increasing amounts of LAB027 dose-dependently decreased their proliferative rates and their viability (Figures 2a and b). At 1 μM, LAB027 decreased CT26 and HT29 proliferation rates by 54% and 57%, respectively (P<0.001 for CT26 and P<0.001 for HT29), and decreased their viability by 31 and 27%, respectively (P<0.001 for CT26 and P<0.05 for HT29). Incubation of NIH3T3 or W138 fibroblasts with increasing amounts of LAB027, dose-dependently decreased their proliferation rates and their viability. At 1 μM LAB027 decreased NIH3T3 and W138 proliferation rates by 28 and 25%, respectively (P<0.001 for NIH3T3 and P<0.001 for W138), and decreased their viability by 20 and 17%, respectively (P<0.05 for NIH3T3 and P<0.05 for W138) (Figures 2a and b). LAB027 was significantly more cytostatic and cytotoxic in CT26 and in HT29 tumor cells lines compared with non-tumoral NIH3T3 and W138 fibroblasts (cytostatic effect of 1 μM LAB027: CT26 versus NIH3T3, P<0.001; CT26 versus W138, P<0.001; HT29 versus NIH3T3, P<0.001; HT29 versus W138, P<0.001; cytotoxic effect of 2 μM LAB027: CT26 versus NIH3T3, P<0.001; CT26 versus W138, P<0.001 ; HT29 versus NIH3T3, P<0.001; HT29 versus W138, P<0.001). The antiproliferative effect of 1 μM LAB027 on CT26, HT29 NIH3T3 and W138 cells was significantly abrogated in vitro by 400 μM N-acetylcysteine (P<0.05; P<0.02; P<0.05; P<0.05, respectively). By contrast, the SOD mimic MnTBAP that produces H2O2 through the dismutation of O2•−, significantly increased the antiproliferative activities of 1 μM LAB027 on CT26 (P<0.05), on HT29 (P<0.02), on NIH3T3 (P<0.05) and on W138 (P<0.05). Incubation of 1 μM LAB027 with the gamma-glutamylcysteine synthetase inhibitor BSO that depletes intracellular GSH, significantly augmented the cytotoxicity of LAB027 in all cell lines (P<0.001). By contrast, blocking catalase by aminotriazol or increasing its intracellular levels by incubation with PEG-catalase had no effects on cell survival or proliferation whatever the cell type considered (Figure 2c).

Figure 2
figure 2

Proliferation rates (a) and cytotoxicity (b) of CT26, HT29, NIH3T3 and W138 cells incubated with various concentrations of LAB027. Results are the mean±S.E.M. of three experiments performed in triplicates. Proliferation (c) of CT26, HT29, NIH3T3 and W138 cell lines following incubation with 1 μM LAB027 alone, or associated with either 400 μM N-acetylcysteine, 400 μM DL-buthionine-(S,R)-sulfoximine (BSO), 200 μM 3-amino-1,2,4-triazole (AT), 50 units/ml catalase PEG (CAT) or with 100 μM MnTBAP. Cellular proliferation was mesured by thymidine incorporation. Error bars represent S.E.M.

Cytotoxic pathways engaged by LAB027

To determine the pathway through which LAB027 promotes tumor cell death, we examined by western blot the cleavage of caspase-3 and caspase-8 proteins 24 h after incubation of HT29 tumor cells with increasing amounts of LAB027. Although the expression of cleaved caspase-8 fragments was dose-dependently upregulated in HT29 cells treated with LAB027, no caspase-3 cleavage was observed in LAB027-treated HT29 cells at any concentration tested (Figure 3a). Incubation of HT29, CT26 or NIH 3T3 cells with either caspase-3-specific inhibitor Ac-DEVD-FMK, or with caspase-8 inhibitor Z-IETD-FMK or with the broad-spectrum caspase inhibitor Z-VAD-FMK along with LAB027 did not modify the cytotoxic effects of LAB027 (Figure 3b), whereas the latter two caspases inhibitors effectively inhibited caspase-8 cleavage at the concentration tested (not shown). Early intracellular events that occur during cell death are associated with mitochondrial changes mediated by members of the Bcl-2 family of proteins, including antiapoptotic Bcl-2, Bcl-xL and pro-apoptotic Bax, Bad proteins. In addition, treatment of HT29 cells with LAB027 triggered an autophagic process (Figure 3) as demonstrated by ATG8 induction and cleavage, and by autophagic vacuolization as demonstrated by acridine orange staining. To clarify the mode of cell death, LAB027-treated HT29 cells were stained with PI and YOPRO-1. After 24-h incubation with 4 μM LAB027, 5%±3 HT29 were positive for YOPRO-1 alone and 33%±7 HT29 were positive for PI alone or with YOPRO-1 showing a necrotic process (Figure 3d). A kinetic analysis of the mode of cell death between 6 and 24 h indicated that LAB027 induces cell death mainly through a necrotic process (Figure 3e).

Figure 3
figure 3

Pro-apoptotic effects of LAB027 (a). HT29 cells were treated or not with increasing concentrations of LAB027 for 24 h. Expressions of cleaved caspase-8, pro-caspase-3 and cleaved caspase-3, Bax, Bad, Bcl-xL, Bcl-2 and ATG8 proteins were analyzed by western blot. Proliferation rates (b) of CT26, HT29 and NIH3T3 cells incubated with various concentrations of LAB027 alone or associated with either 40 μM DEVD-FMK (caspase-3 inhibitor) or 40 μM IETD-FMK (caspase-8 inhibitor) 40 μM ZVAD-FMK (broad-spectrum caspase inhibitor). Results are means±S.E.M. of three independent experiments. (c) Immunofluorescence microscopy of HT29 cells stained with acridine orange and treated for 24 h or not with 4 μM LAB027. An increased number of cells with stained acidic vesicular organelles (orange fluorescence) in 4 μM LAB027 treated cells was observed. (d) Fluorescence-activated cell sorting analysis of cell death. Apoptosis/necrosis was analyzed by flow cytometry using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO-1 and Propidium iodide on HT29 treated (right panel) or not (left panel) with 4 μM LAB027 for 24 h. Necrotic cells are PI positive and YO-PRO-1 positive or negative. Apoptotic cells are PI negative and YO-PRO-1 positive. Viable cells are negative for both dyes. One representative experiment of three is shown. (e) Kinetic apoptosis/necrosis evolution in HT29 cells treated with 4 μM LAB027 alone or with oxaliplatin 0.5 μM

LAB027 synergized with oxaliplatin to trigger ROS production and exert cytostatic effects on colon cancer cell lines in vitro

Co-incubation of CT26 or HT29 cells with 5 or 0.5 μM oxaliplatin and increasing concentrations of LAB027, augmented the production of H2O2 up to 323% (CT26) or 1215% (HT29) at the highest concentration of LAB027 tested. By contrast, co-incubation of mouse NIH3T3 fibroblasts with 5 μM oxaliplatin and LAB027 did not augment the production of H2O2 versus oxaliplatin alone (Figure 4). Moreover, co-incubation of CT26, HT29 or NIH3T3 cells with oxaliplatin (5 μM with CT26 cells and NIH3T3 cells; 0.5 μM with HT29 cells) with increasing concentrations of LAB027 did not augment the production of O2•− versus oxaliplatin alone (Figure 4). Incubation of HT29 colon cancer cells with 1 μM LAB027 along with 0.5 μM oxaliplatin significantly increased the antiproliferative potential of LAB027 by 31% (P<0.05) and the antiproliferative potential of oxaliplatin by 23% (P<0.05). By contrast, incubation of mouse NIH3T3 fibroblasts with 1 μM LAB027 along with 5 μM oxaliplatin did not significantly modify the antiproliferative potential of LAB027 (P=0.68) or of oxaliplatin (P=0.85) administered alone (Figure 5). A kinetic analysis of the mode of cell death induced by co-incubation of LAB027 with oxaliplatin was performed on HT29 cells. The percentages of necrotic cells among apoptotic+necrotic cells were 81%, 84% and 84% after 6, 12 and 24 h incubation, respectively, with LAB027 alone compared with 60, 80 and 83% after 6, 12 and 24 h incubation, respectively, with oxaliplatin+LAB027 (Figure 3e).

Figure 4
figure 4

Production of hydrogen peroxide (a) and superoxide anions (b) by oxaliplatin-treated CT26, HT29 and NIH3T3 cells incubated with increasing concentrations of LAB027. Differences (delta) in H2O2 production (a) and in O2•− production (b) between oxaliplatin-treated cells and untreated cells are shown. Results are the mean±S.E.M. of three experiments performed in triplicate

Figure 5
figure 5

Proliferation (a) of CT26, HT29 and NIH3T3 cells treated with LAB027 alone or in association with oxaliplatin. Error bars represent S.E.M. LAB027 concentration was 1 μM for all the cells tested. Intracellular levels of reduced glutathione (b) in CT26 cells, HT29 cells and NIH3T3 cells treated with various concentrations of LAB027. Results are the mean±S.E.M. of three experiments each performed in triplicate. Proliferation (c) of CT26, HT29 and NIH 3T3 cell lines following incubation with 1 μM LAB027 plus oxaliplatin alone, or associated with either 400 μM N-acetylcysteine, 400 μM DL-buthionine-[S,R]-sulfoximine (BSO), 200 μM 3-amino-1,2,4-triazole (AT), 50 units/ml catalase PEG (CAT) or with 100 μM MnTBAP. Oxaliplatin concentration was respectively 5, 0.5 and 5 μM for CT26, HT29 and NIH3T3 cells. Cellular proliferation was mesured by thymidine incorporation. Error bars represent S.E.M.

Imbalance of GSH metabolism in tumor cells

In normal NIH3T3 cells, increasing the oxidative stress by incubation with 4 μM LAB027 augmented intracellular GSH levels by 311% (Figure 5). Adding 5 μM oxaliplatin to LAB027, further increased GSH synthesis by 80%. Interestingly, incubating CT26 or HT29 colon cancer cells with LAB027 alone or in association with oxaliplatin failed to increase GSH synthesis (Figure 5). In order to determine whether this phenomenon was linked to a differential effect of LAB027 on GSH efflux in normal and tumor cells, we measured the GSH released from CT26, HT29 or NIH3T3 upon incubation with increasing concentrations of LAB027 alone or in association with oxaliplatin. Increasing concentrations of LAB027 significantly induced GSH release, but no difference was observed between normal and tumor cells (Supplementary Figure 3).

Co-incubation of oxaliplatin and LAB027 with the glutathione reductase inhibitor BSO that depletes the cells in reduced glutathione, significantly increased the antiproliferative potential of oxaliplatin+LAB027 in all cell lines (P<0.001). In addition, incubation with NAC, a precursor of GSH significantly increased cell proliferation (P<0.001 for HT29 and CT26 and P<0.05 for 3T3). By contrast, blocking catalase with aminotriazol or increasing its intracellular levels with PEG-catalase had no effect on cell survival and proliferation whatever the cell type considered (Figure 5).

Effect of LAB027 on the growth of CT26 tumor cells implanted in mice treated with oxaliplatin

To determine whether LAB027 could inhibit tumor growth in vivo, we treated mice bearing CT26 tumors with either oxaliplatin or LAB027, or with the association of oxaliplatin and LAB027 (Figure 6a). Mice treated with oxaliplatin alone developed significantly smaller tumors than untreated mice (–58%, P<0.001 versus untreated mice on day 27). LAB027 also inhibited tumor growth when administered alone (−25% on day 27, P<0.05 versus untreated mice). Mice treated simultaneously with LAB027 and oxaliplatin had significantly smaller tumors than untreated mice on day 27 (−75%, P<0.001). Furthermore, the administration of LAB027 amplified the antitumor effect observed with oxaliplatin alone (−41% versus oxaliplatin-treated mice, P<0.05 on day 27). In order to determine whether the immune system has a role in the antitumoral effects induced by LAB027 alone or in combination with oxaliplatin, we performed the same experiment using immunodeficient SCID mice. In SCID mice implanted with CT26, LAB027 inhibited tumor growth when administered alone (−24% at day 27, P<0.05 versus untreated mice). SCID mice treated simultaneously with LAB027 and oxaliplatin had significantly smaller tumors than untreated mice at day 27 (−78%, P<0.001). The administration of LAB027 amplified the antitumor effect observed with oxaliplatin alone (−56% versus oxaliplatin-treated mice, P<0.05 at day 27).

Figure 6
figure 6

Tumor growth following implantation of 2 × 106 CT26 cells in BALB/c mice (a) or in SCID mice (b). Mice were treated as described in the ‘Materials and Methods section’ (seven mice/group). Biological toxicity (c): leukocytes, neutrophils and platelets count in the blood of mice on day 14, following two i.v. injections of oxaliplatin (10 mg/kg) on days 1 and 7 and two injections of LAB027 (10 mg/kg) on days 1 and 7. Survival rate of mice (d) following intraperitoneal injection of 107 E. coli in BALB/c mice. Mice were treated as described in the ‘Material and Method section’ (10 mice/group)

In vivo effects of LAB027 on the toxicity of oxaliplatin and on the susceptibility of mice to bacterial infection

We next investigated the effects of LAB027 on the toxicity of oxaliplatin in BALB/c mice. The toxicity on liver was evaluated by measuring serum concentrations of ALAT, LDH and alkaline phosphatases. Kidney function was assessed on serum BUN and creatinin (Supplementary Figure 4). None of these parameters were altered in the serum of mice treated with oxaliplatin or LAB027 alone, or in the serum of mice treated with both LAB027 and oxaliplatin. After 14 days the first injection of oxaliplatin, a significant decrease in the absolute numbers of peripheral leukocytes, neutrophils and platelets was observed versus control mice treated with PBS (Figure 6b). LAB027 alone had no hematological toxicity compared with untreated control mice. Furthermore, the administration of LAB027 in association with oxaliplatin significantly decreased the hematological toxicity of oxaliplatin. Indeed, the counts of peripheral leukocytes (P=0.027), neutrophils (P=0.008) and platelets (P=0.035) in the blood of mice treated with LAB027 and oxaliplatin were higher than in the blood of mice treated with oxaliplatin alone. Because bacterial infection is the main complication of neutropenia, the susceptibility of mice to intraperitoneal inoculation of E. coli was tested following the administration of oxaliplatin alone or in association with LAB027 (Figure 6c). In all, 40% of control mice treated with PBS survived 28 h after E. coli inoculation. However, no animal treated with oxaliplatin before E. coli inoculation, survived more than 22 h following the bacterial challenge. In contrast, 55% of mice treated with oxaliplatin and LAB027 survived 28 h following intraperitoneal inoculation of E. coli (P=0.044).

Discussion

This study provides pre-clinical evidence that the organotelluride catalyst LAB027, a prototypical member of family of the ‘sensor/effector’ multifunctional redox catalyst agents, is a new candidate molecule to treat colon cancer either alone or in combination with oxaliplatin.

Previous experiments have shown that ‘sensor/effector’ agents can act as cytotoxic molecules in vitro.25 These agents are catalytic molecules that recognize a particular intracellular state of cells – such as an oxidative stress – and selectively kill the cells that are under this state. In our hands, tumor cells display an elevated basal level of ROS that is amplified by LAB027 to generate a lethal oxidative burst. The most likely explanation for this amplifying effect is an association of oxygen radical and H2O2 formation at the quinone and oxidation of cellular thiols catalyzed by tellurium in the presence of H2O2. Those chemical reactions are likely to increase the oxidative stress in tumor cells with a high initial level of H2O2, and ultimately push those cells over a critical ‘life and death’ (redox) threshold.24 This also accounts for the differences of toxicity of the LAB027 compound in tumor and normal cells. Moreover in tumor cells, the levels of antioxidant enzyme activities are low and not modified by LAB027. In normal cells, the basal levels of antioxidant enzyme activities are higher and are still raised by LAB027, thus preventing cell death.9 The differences between normal and tumor cells in terms of basal oxidative stress and antioxidant enzyme levels have already been used to selectively kill tumor cells in vitro. Thus, SOD mimics favor the dismutation of superoxide anions overproduced by tumor cells, and consecutively raise the production of H2O2 up to a cytotoxic level that is not reached in normal cells with low basal levels of H2O2.9, 16 The mechanism of action of LAB027 is different; it induces by itself an oxidative stress that is amplified by the pre-existing oxidative stress in tumor cells. H2O2 and not O2•− has a key role in the death induced by LAB027, as N-acetylcysteine but not the SOD mimic MnTBAP significantly decreases LAB027 toxicity. Incubation of LAB027 with the gamma-glutamylcysteine synthetase inhibitor BSO that depletes the cells in reduced glutathione significantly increases the cytotoxicity of LAB027 in all cell lines. By contrast, blocking catalase with aminotriazol or increasing its intracellular levels with PEG-catalase, has no effects on cell survival and proliferation whatever the cell type considered. These results indicate that H2O2-mediated LAB027 toxicity is controlled by the glutathione pathway rather than by the catalase pathway, as already observed with several chemotherapeutic drugs.

The role of ROS in tumor cell death induced by LAB027 is emphasized by the effects of the compound on the mitochondrial release of pro-apoptotic molecules. Members of the Bcl-2 family control the permeability of the mitochondrial outer membrane to various pro-apoptotic proteins.26 In our hands, LAB027 exerts its powerful cytotoxic effect by inhibiting the expression of Bcl-2 and Bcl-xL, two antiapoptotic molecules, and increasing the expression of Bax and Bad, two pro-apoptotic molecules. This alteration in the mitochondrial membrane composition leads to the mitochondrial collapse, but is not followed by caspase-3 cleavage as usually observed during apoptosis.26 Those results are in line with the ineffectiveness of caspase-3 inhibitors to abrogate LAB027-induced cell death and with the necrotic mode of death induced by LAB027 as demonstrated by YOPRO-1/PI staining of LAB027-treated cells. ROS have been reported to induce cell death by necrosis and/or apoptosis depending on the rate of ROS production, the duration of the oxidative burst and the cell type considered.27 In our hands, LAB027 triggers a caspase-independent, ROS-mediated cell death by necrosis. LAB027 also induces an autophagic process as demonstrated by ATG-8 induction and cleavage, and by the phenomenon of vacuolization. Indeed, H2O2 can trigger the formation of autophagosomes through the oxidation of a critical cysteine residue on HsAtg4A. The resulting inactivation of HsAtg4A leads to the accumulation of Atg8-PE on the phagophore membrane and to the formation of autophagosomes.28 However, it is not clear whether this mechanism is involved in cell death, or counteracts the toxic effects of LAB027 and favours cell survival by removal of harmful protein aggregates and damaged mitochondria, which are sources of altered proteins.29

The chemotherapeutic treatment of colon cancer usually requires the use of several drugs. Oxaliplatin is one of the most frequently used.30, 31 In our hands, the augmentation of the antiproliferative effects of oxaliplatin on colon cancer cells when associated with LAB027, is linked to the increased intracellular concentration of H2O2. However, a similar increase in H2O2 concentration is observed in NIH3T3 fibroblasts treated by oxaliplatin and LAB027, and no antiproliferative effect is observed. This is explained by the increase in GSH levels that occurs in normal 3T3 fibroblasts but not in colon cancer cells. Because of its role in H2O2 detoxification, the glutathione pathway represents a major component of cellular defense against cytotoxic drugs as oxaliplatin These results are in agreement with the effect of LAB027 on glutathione reductase levels, an enzyme that restores the pool of reduced GSH and whose level is increased in normal but not in tumor cells. The role of glutathione reductase is also emphasized by the synergistic effect of the gamma-glutamylcysteine synthetase inhibitor BSO with LAB027 and oxaliplatin on tumor cells. The effect is still more dramatic on normal cells.

Thus, our data confirm that oxaliplatin exerts its anti-tumoral effects at least partly through the induction of an oxidative stress and emphasizes the role of reduced glutathione in the control of oxaliplatin cytotoxicity.32 In tumor cells, high levels of reduced glutathione, the cofactor of glutathione peroxidase, have been associated with a multidrug resistance phenotype.19 Compounds that target such protective mechanisms could enhance the activity of anticancer agents and reverse the multidrug resistance phenotype. For example, BSO, an inhibitor of glutathione synthesis, can increase the cytotoxicity of melphalan by preventing glutathione peroxidase activity and increasing H2O2 levels.20 However, BSO depletes glutathione in both normal and cancer cells, which increases melphalan's hematological toxicity and prevents any enhancement of the therapeutic index of this anticancer agent.33, 34 Conversely, N-acetylcysteine protects normal cells from the cytotoxic effects of anticancer drugs by preventing the elevation of intracellular H2O2 through its reductive properties,9 but abrogates the effectiveness of chemotherapeutic drugs. We have already observed that, as LAB027, mangafodipir prevents the hematological toxicity of oxaliplatin, but enhances its antitumoral activity against colon cancer both in vitro and in vivo.16 The differential cytotoxic effects of mangafodipir on normal and cancer cells also result from differences in the redox status of those cells. Mangafodipir induces an overproduction of H2O2 through the dismutation of superoxide anions, but decreases the toxicity of H2O2 in normal cells through its glutathione reductase-like activity. Both mangafodipir and LAB027 increase the therapeutic index of oxaliplatin in vitro and in vivo, prevent hematologic toxicity and the death of animals injected with a lethal concentration of bacteria. Moreover, arsenic trioxide, one of the prototypical ‘sensor/effector’ chemotherapeutic agents, exerts its antitumoral activity through the induction of an oxidative burst without inducing leucopenia.15

Only a small number of studies have previously explored the antitumoral role of tellurium compounds. Only one tellurium-based compound, AS101, has been tested so far. AS101 (ammonium trichloro (dioxoethylene-O,O′) tellurate), a tellurium (IV) compound, is endowed with antitumor properties in several murine models.35, 36 Phase I clinical trials on patients with advanced cancer treated with AS101 have shown an increased production and secretion of a variety of cytokines, leading to a clear dominance of Th1 responses with a concurrent decrease in Th2 responses.37 The predominance of Th1 responses is related to the antitumor activity of AS101. In addition, AS101, as a tellurium (IV) compound, can interact with thiols to form a Te-S bond. The ability of AS101 to interact with cysteine residue can alter protein functions,38 increase intracellular ROS and favor the induction of apoptosis.39 However, all the activities of AS101 are associated with little toxicity, and this drug is safe for clinical applications.40

The present study provides the first pre-clinical evidence that the organotelluride catalyst, LAB027 is a potential drug to treat colon cancer either alone or in combination with oxaliplatin through a mechanism that involves mainly necrosis. Tumor cell death is associated with the overproduction of intracellular ROS but with no elevation of reduced glutathione levels as observed in normal cells. This difference may explain the limited kidney, liver and hematological toxicity of this compound in vivo. Finally, this report shows for the first time that ‘sensor/effector’ pro-oxidative agents could represent effective and safe options to treat patients with colon cancer.

Materials and Methods

Animals

BALB/c and BALB/c SCID female mice between 6 and 8 weeks of age were used in all experiments (Harlan, Gannat, France). All mice were housed in autoclaved cages with sterile food and water. Animals received humane care in compliance with institutional guidelines.

LAB027 synthesis

The synthesis of LAB027 is described in detail in Supplementary files (Supplementary data and Supplementary Figure 1).

Chemicals, cell lines and culture

All chemicals were from Sigma (Saint Quentin Fallavier, France) except for oxaliplatin (Eloxatine, Sanofi-Pharma, Paris, France). CT26 (mouse colon carcinoma), HT29 (human colon carcinoma), NIH 3T3 (mouse fibroblast) and W138 (human lung fibroblast) were obtained from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in DMEM/glutamax-I supplemented with 10% heat-inactivated FCS and antibiotics (Life Technologies, Cergy Pontoise, France). All cell lines were routinely tested for Mycoplasma infection.

Cellular production of O2•− and H2O2

Cells (2 × 104 cells/well) were seeded in 96-well plates (Costar, Corning, Inc., Corning, NY) and incubated for 48 h in culture medium alone or added with various concentrations of LAB027 alone or in combination with oxaliplatin. Levels of intracellular superoxide anion O2•− and H2O2 were assessed spectrofluorometrically (Packard Bioscience, Boston, MA, USA) by oxidation of dihydroethidium (DHE) (Molecular Probes, Leiden, The Netherlands) and of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes), respectively, as previously described.16 The levels of O2•− or H2O2 were calculated in each sample as follows: ROS rate (arbitrary units/min/106 cells)=(fluorescence intensity (arbitrary units) at T60 minutes−fluorescence intensity (arbitrary units) at T0)/60 min/number of viable cells as measured by the crystal violet assay.

Determination of enzymatic activities

The SOD activity in tumor or normal cells was evaluated by the method of Beauchamp and Fridovich30 and expressed as units/106 cells. The catalase activity in tumor or normal cells was determined according to Aebi41 and expressed as units/106 cells. The glutathione reductase assay was performed by the method of Carlberg and Mannervik42 and expressed as nmol/min/106 cells. Levels of extracellular GSH were measured by the method of Baker et al.43 and expressed as nmol/106 cells.

In vitro cell proliferation and viability assays

CT26, HT29, NIH 3T3 or W138 cells (2 × 104 cells/well) were seeded into 96-well plates and incubated for 48 h in complete DMEM medium with varying amounts of LAB027 alone or with oxaliplatin. Cell proliferation was determined by pulsing the cells with [3H] thymidine (1 μCi/well) during the last 16 h of culture. Cell viability was evaluated by the crystal violet assay.16 Results are expressed as percentages of viable cells compared with untreated cells (that have 100% viability).

Levels of intracellular and extracellular reduced glutathione

The levels of intracellular glutathione were assessed spectrofluorometrically by monochlorobimane staining.44 Briefly, tumor cells (2 × 104/well) or normal NIH3T3 (2 × 104/well) were seeded in 96-well plates and incubated for 48 h in complete medium with LAB027 alone or in combination with oxaliplatin at concentrations described in figure legends. Cells were washed once with PBS and then incubated with 50 μM monochlorobimane in PBS. The fluorescence intensity was measured after 15 min at 37°C. Excitation and emission wavelengths were 380 and 485 nm, respectively. The intracellular glutathione level was expressed as arbitrary units of fluorescence intensity/number of adherent cells as measured by the crystal violet assay. Extracellular reduced glutathione was assayed in culture supernatants using the method of Baker et al.43

Immunoblotting of cell lysates

HT29 cells were treated or not with 1, 2 or 4 μM LAB027 for 24 h then lysed in ice-cold RIPA.9 Protein (50 μg) extracts were analyzed by immunoblotting after 10–15% SDS-PAGE using of rabbit polyclonal anti-mouse pro- and active caspase-3 (1 : 1500, Abcam 47131, Paris, France), rabbit polyclonal anti-mouse caspase-8, mouse polyclonal anti-mouse Bcl-2, Bcl-xL and Bax primary antibodies (1 : 200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bad primary antibody (1 : 100, Santa Cruz Biotechnology), ATG-8 primary antibody (1 : 500, Sigma-Aldrich, Saint-Quentin Fallavier, France) and mouse monoclonal anti-β-actin (1 : 50 000, Sigma). After washing, membranes were incubated with a 1 : 1000 goat anti-rabbit or goat anti-mouse secondary antibody (Sigma-Aldrich). The membranes were developed in Super Signal West Pico Chemiluminescent Substrate (Pierce, CO, USA). Images of blots were obtained using Fujifilm LAS3000 digital imager analyzed with Fujifilm Multi-Gauge Imaging software (Fujifilm, Tokyo, Japan).

Modulation of the catalase and glutathione reductase pathways

HT29, CT26, NIH 3T3 or W138 cells (2 × 104 cells/well) were pre-incubated for 2 h with either 400 μmol/l N-acetyl cysteine (NAC), or with 50 units/ml PEG-catalase, or with 200 μmol/l aminotriazol (ATZ, a catalase inhibitor) or with 400 μmol/l L-buthionine-SR-sulfoximine (BSO, a gamma-glutamylcysteine synthetase inhibitor). Those concentrations were chosen because they significantly modulated either reduced glutathione concentration (BSO, NAC) or catalase activity (PEG-catalase, ATZ) (not shown). The cells were then incubated with 1 μM LAB027 alone or in combination with oxaliplatin for 48 h. Cell proliferation was assayed by pulsing the cells with [3H] thymidine (1 μCi/well) during the last 16 h of culture.

Study of caspase inhibition

HT29 cells (2 × 104 cells/well) were pre-incubated for 2 h with either 40 μM caspase-3-specific inhibitor Ac-DEVD-FMK or with 40 μM caspase-8 inhibitor Z-IETD-FMK, or with the broad-spectrum 40 μM caspase inhibitor Z-VAD-FMK. The cells were then incubated with LAB027 for 48 h. Cell viability was evaluated by the crystal violet assay. Results are expressed as percentages of viable cells compared with untreated cells (100% viability).

Fluorescence-activated cell sorting analysis of cell death

Apoptosis and necrosis were analyzed by the fluorescence-activated cell sorting (FACS) Canto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA), using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO-1 and Propidium iodide (PI) for Flow Cytometry (Invitrogen, Cergy Pontoise, France) under the manufacturer's recommendations. Briefly, 5 × 105 HT29 cells were incubated with 4 μM LAB027 for a period of time indicated in the legends of figures. After the incubation period, cells were collected and stained for 30 min on ice with 1.5 μM PI and 0.1 μM YO-PRO-1. Cells were washed two times and analyzed by flow cytometry.

Acridine orange staining for autophagy detection

Acridine orange (Sigma) staining was performed according to Kanzawa et al.45 Briefly, HT29 cells were treated or not for 24 h with 4 μM LAB027. After the incubation period, cells were washed twice with phosphate-buffered saline and stained with 1 μM acridine orange for 15 min at 37°C. Cells were washed and analyzed by fluorescence microscopy using 490-nm band-pass blue excitation filters and a 515-nm long-pass barrier filter.

In vivo antitumor activity of LAB027

CT26 (2 × 106) cells were injected subcutaneously into the back of BALB/c or BALB/c SCID mice. When the tumors reached a mean size of 200–500 mm3, mice were randomized (day 10) in each experimental and control groups depending on tumor size, in order to start the treatment with a similar mean size in each group. One group of seven mice was treated by intraperitoneal oxaliplatin (10 mg/kg/week) starting on day 10. One group of seven mice was treated by intravenous LAB027 (10 mg/kg/week) starting on day 10. One group of seven mice was treated with intravenous LAB027 (10 mg/kg/week) and intraperitoneal oxaliplatin (10 mg/kg/week) starting on day 10. One control group of seven mice was injected with PBS. Tumor size was measured with a calliper rule every 2 days. Tumor volume was calculated as follows: TV (mm3)=(L × W2)/2, where L is the longest and W the shortest radius of the tumor in millimeters. Results were expressed as means of tumor volumes±S.E.M. (n=7 in each group).

In vivo analysis of blood, liver and kidney toxicity

Mice were injected with PBS alone or intraperitoneal, oxaliplatin alone, or intravenous LAB027 alone or intraperitoneal oxaliplatin in association with intravenous LAB027 at days 0 and 7. Five mice were treated in each group. After 14 days of the first injection, mice were killed by cervical dislocation. Blood samples were then collected from each mouse. Leukocytes, neutrophils and platelets were enumerated using a Malassez cell after hypotonic red blood cell lysis. Liver enzymes ALAT, LDH, alkaline phosphatases, BUN and creatinine were assayed using a multiparametric analyzer (Hitachi 747, Roche Diagnostics, Meylan, France).

Susceptibility of mice to bacterial infection

BALB/c female mice were injected with vehicle alone (PBS), or intraperitoneal oxaliplatin alone, or intravenous LAB027 alone or intraperitoneal oxaliplatin and intravenous LAB027 days 0 and seven. Five mice were treated in each group. After 14 days of the first injection, mice were injected i.p with a lethal dose of 107 E. coli on day 9. The survival rate of mice was evaluated during 24 h following E. coli injection. A total of 10 mice were treated in each group in two independent experiments.

Statistical analysis

The statistical significance of differences between experimental treated groups and untreated controls was analyzed by Student's t-test for comparison of means. A level of P<0.05 was accepted as significant. *P<0.05; ***P<0.01 versus controls.