Oxaliplatin neurotoxicity involves peroxisome alterations. PPARγ agonism as preventive pharmacological approach

Abstract

The development of neuropathic syndromes is an important, dose limiting side effect of anticancer agents like platinum derivates, taxanes and vinca alkaloids. The causes of neurotoxicity are still unclear but the impairment of the oxidative equilibrium is strictly related to pain. Two intracellular organelles, mitochondria and peroxisomes cooperate to the maintaining of the redox cellular state. Whereas a relationship between chemotherapy-dependent mitochondrial alteration and neuropathy has been established, the role of peroxisome is poor explored. In order to study the mechanisms of oxaliplatin-induced neurotoxicity, peroxisomal involvement was evaluated in vitro and in vivo. In primary rat astrocyte cell culture, oxaliplatin (10 µM for 48 h or 1 µM for 5 days) increased the number of peroxisomes, nevertheless expression and functionality of catalase, the most important antioxidant defense enzyme in mammalian peroxisomes, were significantly reduced. Five day incubation with the selective Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) antagonist G3335 (30 µM) induced a similar peroxisomal impairment suggesting a relationship between PPARγ signaling and oxaliplatin neurotoxicity. The PPARγ agonist rosiglitazone (10 µM) reduced the harmful effects induced both by G3335 and oxaliplatin. In vivo, in a rat model of oxaliplatin induced neuropathy, a repeated treatment with rosiglitazone (3 and 10 mg kg(-1) per os) significantly reduced neuropathic pain evoked by noxious (Paw pressure test) and non-noxious (Cold plate test) stimuli. The behavioral effect paralleled with the prevention of catalase impairment induced by oxaliplatin in dorsal root ganglia. In the spinal cord, catalase protection was showed by the lower rosiglitazone dosage without effect on the astrocyte density increase induced by oxaliplatin. Rosiglitazone did not alter the oxaliplatin-induced mortality of the human colon cancer cell line HT-29. These results highlight the role of peroxisomes in oxaliplatin-dependent nervous damage and suggest PPARγ stimulation as a candidate to counteract oxaliplatin neurotoxicity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Catalase immunostaining in primary astrocytes.

Cells (5·104 cells/well) were incubated for 5 days with 10 µM rosiglitazone (B), 1 µM oxaliplatin (C), 1 µM oxaliplatin+10 µM rosiglitazone (D), 30 µM G3335 (E), negative control (F) in comparison to control condition (A). Representative images are shown in the left panel. Scale bar 50 µm. The measurements of the number of peroxisomes/µm2 and the catalase optical density per number of peroxisomes are shown in the upper and lower graphs, respectively. *P<0.01 vs control; ∧P<0.01 vs 1 µM oxaliplatin.

Figure 2. Expression and activity of catalase in astrocyte cell culture.

Astrocytes (5·105 cells/well) were treated with the PPARγ antagonist G3335 (30 µM) or with oxaliplatin (1 µM) in the absence or in the presence of the PPARγ agonist rosiglitazone (10 µM). Expression and activity were measured after 48 h- (A and B, respectively) or 5 day-treatment (C and D, respectively). GAPDH normalization was performed for each sample. Values are expressed as the mean ± S.E.M. percent of control of three experiments. Control condition was arbitrarily set as 100%. *P<0.05 vs control; ∧P<0.05 vs 1 µM oxaliplatin.

Figure 3. Pain threshold measurements.

A) Noxious stimulus, Paw-pressure test. Rats were daily intraperitoneally treated with 2.4 mg kg−1 oxaliplatin (dissolved in 5% glucose). Rosiglitazone (3 and 10 mg kg−1, suspended in CMC) was per os daily administered starting from the first day of oxaliplatin administration. B) Non-noxious stimulus, Cold plate test. The response to a thermal stimulus was evaluated by cold plate test measuring the latency (seconds) to pain-related behaviors (lifting or licking of the paw). Control animals were treated with vehicles. Behavioral measures were performed on day 7, 14 and 21, 24 h after the last treatment. Each value represents the mean of 10 rats per group, performed in 2 different experimental set. *P<0.01 vs vehicle + vehicle (control); ∧P<0.01 vs oxaliplatin + vehicle.

Figure 4. Motor coordination in oxaliplatin-treated rats.

The integrity of the animals’ motor coordination was assessed using a rota-rod apparatus. Rats were placed on a rotating rod (10 rpm) for a maximum of 10 minutes (600 seconds). The number of falls (A) and the time spent in the balance (B) during 10 minutes were counted. Treatments (oxaliplatin 2.4 mg kg−1 i.p. and rosiglitazone 3 and 10 mg kg−1 p.o.) were performed daily. Motor coordination was evaluated on day 21, 24 h after the last treatment. Each value represents the mean of 10 rats per group, performed in 2 different experimental set. *P<0.01 vs vehicle + vehicle (control); ∧P<0.05 vs oxaliplatin + vehicle.

Figure 5. Expression and activity of catalase in the nervous tissue of oxaliplatin-treated animals.

On day 21, dorsal root ganglia (DRG) and spinal cord were analyzed to measure both expression and activity of catalase. Densitometric analysis and representative Western blot of catalase expression in DRG (A) and spinal cord (C) are shown. GAPDH normalization was performed for each sample. Catalase enzymatic activity measurements in DRG (B) and spinal cord (D). Values are expressed as the mean ± S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of *P

Figure 6. Levels of carbonylated proteins in the spinal cord of oxaliplatin-treated rats.

At 21th day, the lumbar tract of the spinal cord was explanted and analyzed to measure protein oxidation. Densitometric analysis (top panel) and representative Western blot (lower panel) are shown. β-actin normalization was performed for each sample. Values are expressed as the mean ± S.E.M. percent of control of 10 rats per group, performed in 2 different experimental set. Each value represents the mean of *P<0.05 vs vehicle + vehicle; ∧P<0.05 vs oxaliplatin + vehicle.

Figure 7. Glial profile in spinal cord scored with GFAP-positive cells in the dorsal horn of the lumbar tract.

Transverse sections of spinal cord imaged with 20X objective of A) vehicle + vehicle, B) oxaliplatin + vehicle, C) and D) oxaliplatin + rosiglitazone 3 and 10 mg kg−1, respectively. Scale bar 50 µm. In the lower panel quantitative analysis of cellular density is shown. Each value represents the mean ± S.E.M. of 10 rats per group, performed in 2 different experimental sets. *P<0.05 vs vehicle + vehicle; ∧P<0.05 vs oxaliplatin + vehicle.