Preventive action of benztropine on platinum-induced peripheral neuropathies and tumor growth

Abstract

The endogenous cholinergic system plays a key role in neuronal cells, by suppressing neurite outgrowth and myelination and, in some cancer cells, favoring tumor growth. Platinum compounds are widely used as part of first line conventional cancer chemotherapy; their efficacy is however limited by peripheral neuropathy as a major side-effect. In a multiple sclerosis mouse model, benztropine, that also acts as an anti-histamine and a dopamine re-uptake inhibitor, induced the differentiation of oligodendrocytes through M1 and M3 muscarinic receptors and enhanced re-myelination. We have evaluated whether benztropine can increase anti-tumoral efficacy of oxaliplatin, while preventing its neurotoxicity.We showed that benztropine improves acute and chronic clinical symptoms of oxaliplatin-induced peripheral neuropathies in mice. Sensory alterations detected by electrophysiology in oxaliplatin-treated mice were consistent with a decreased nerve conduction velocity and membrane hyperexcitability due to alterations in the density and/or functioning of both sodium and potassium channels, confirmed by action potential analysis from ex-vivo cultures of mouse dorsal root ganglion sensory neurons using whole-cell patch-clamp. These alterations were all prevented by benztropine. In oxaliplatin-treated mice, MBP expression, confocal and electronic microscopy of the sciatic nerves revealed a demyelination and confirmed the alteration of the myelinated axons morphology when compared to animals injected with oxaliplatin plus benztropine. Benztropine also prevented the decrease in neuronal density in the paws of mice injected with oxaliplatin. The neuroprotection conferred by benztropine against chemotherapeutic drugs was associated with a lower expression of inflammatory cytokines and extended to diabetic-induced peripheral neuropathy in mice.Mice receiving benztropine alone presented a lower tumor growth when compared to untreated animals and synergized the anti-tumoral effect of oxaliplatin, a phenomenon explained at least in part by benztropine-induced ROS imbalance in tumor cells.This report shows that blocking muscarinic receptors with benztropine prevents peripheral neuropathies and increases the therapeutic index of oxaliplatin. These results can be rapidly transposable to patients as benztropine is currently indicated in Parkinson's disease in the United States.

Keywords: Benztropine; Muscarinic receptors; Myelin; Oxaliplatin; Peripheral neuropathies.

Fig. 1

In vivo effects of oxaliplatin and benztropine on acute oxaliplatin peripheral neuropathy. Evaluation of hyperalgesia required two 5-day cycles of daily oxaliplatin (3 mg/kg). Control mice received either oxaliplatin or vehicle alone. Cold hyperalgesia was evaluated using a cold plate set at + 2 °C. Data are expressed as means ± SEM of 7 different mice under each condition. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle

Fig. 2

In vivo effects of oxaliplatin and benztropine on chronic oxaliplatin peripheral neuropathy. a von Frey test and b cold-plate hypoesthesia test. Experimental mice received oxaliplatin (10 mg/kg) weekly and benztropine (10 mg/kg) daily for 6 weeks. Control mice received either oxaliplatin or vehicle alone. Both the von Frey and the cold-plate tests were performed on a weekly basis. Data are expressed as means ± SEM of 8 different mice under each condition. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle

Fig. 3

In vivo effects of oxaliplatin and benztropine on diabetes-induced peripheral neuropathies. a von Frey test and b hot-plate test. Experimental diabetic mice received benztropine (10 mg/kg) daily for 6 weeks. Control mice received either benztropine or vehicle alone. Both, the von Frey as well as the hot-plate tests were performed on a weekly basis. Data are expressed as means ± SEM of 8 different mice under each condition. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle

Fig. 4

In vivo effects of oxaliplatin and benztropine on mouse sensory excitability variables. Histograms of mean values ± SD of maximal CNAP peak amplitude (upper panel), stimulus intensity necessary to evoke 50% of maximal CNAP amplitude (middle panel), and latency (lower panel), determined from recordings at the tail base in response to caudal nerve stimulation in mice treated for 6 weeks with vehicle (n = 9), oxaliplatin (n = 10), oxaliplatin plus benztropine (n = 10) or benztropine alone (n = 10). *p < 0.05, **p < 0.01 versus vehicle

Fig. 5

In vitro effects of oxaliplatin, associated or not with benztropine, on excitability of mouse DRG sensory neurons, using whole-cell patch-clamp technique. Resting membrane potential (a), and action potential peak amplitude (b), rise time (c) and decay time (d) measured from recordings performed on DRG neurons before (control) and 10–20 min after addition of first 25–50 μM oxaliplatin (O) and then 25–50 μM oxaliplatin plus 10 μM benztropine (B) to the standard physiological medium. Mean ± SD of 5–18 neurons. *p = 0.010–0.014, **p = 0.002–0.005 and ***p < 0.001 versus control

Fig. 6

Ex vivo effects of oxaliplatin and benztropine on the morphology of myelinated axons isolated from mouse sciatic nerves, using confocal microscopy. Representations of nodal length, diameter and volume, as functions of internodal diameter, of myelinated axons isolated from mice (n = 4 in each group) injected with vehicle (black closed circles, n = 137), oxaliplatin (red closed circles, n = 206), oxaliplatin plus benztropine (green closed circles, n = 150) or benztropine alone (blue closed circles, n = 160) for 6 weeks. The curves represent the linear (upper and middle panels) or non-linear (lower panels) fits of data points with R² (correlation coefficients) between 0.441 and 0.877. In left panels, the arrows underline the effects of oxaliplatin

Fig. 7

Effect of benztropine on demyelination and axonal atrophy in oxaliplatin-treated mice. a Representative images of EM micrographs of ultrathin cross-sections of sciatic nerves from vehicle, oxaliplatin, benztropine and oxaliplatin plus benztropine animals at 6 weeks. b Quantification of myelin state though g-ratio analysis reported to axonal caliber. At least 200 axons per animal (n = 2–3) were analyzed. The curves are linear regression fits of data points. *p < 0.05, **p < 0.01

Fig. 8

Effect of benztropine cutaneous nerve fiber density reduction induced by oxaliplatin. a Representative images of staining with PGP9.5 of cutaneous nerve fibers. b Analysis of cutaneous nerve fiber density from paw skin samples (6 μm) of mice treated for 6 weeks with vehicle, oxaliplatin, oxaliplatin plus benztropine or benztropine alone. Mean ± SEM of 8 mice. *p < 0.05, **p < 0.01. NS: non-significant. Scale bar = 50 μm

Fig. 9

Effect of benztropine on MBP expression in the sciatic nerves of oxaliplatin-treated mice. Western blot analyses of total protein lysates from brain sciatic nerves of control, and treated mice. a panel shows detection of MBP and b panel shows anti-β-actin for loading control. NS: non-significant

Fig. 10

Sera inflammatory markers. a ELISA-quantified levels of IL-6 in sera from mice treated for 6 weeks with vehicle, oxaliplatin, oxaliplatin plus benztropine or benztropine alone. b ELISA-quantified levels of TNF-α in sera from mice treated for 6 weeks with vehicle, oxaliplatin, oxaliplatin plus /benztropine or benztropine alone. Data are mean ± SEM of 8 mice. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle. NS: non-significant

Fig. 11

Benztropine prevents tumor growth and displays a synergistic antitumoral effect when associated with oxaliplatin in an ectopic model of colorectal cancer. Tumor size in mice injected subcutaneously into the back with 106 CT26 cells and treated with vehicle, oxaliplatin, oxaliplatin associated with benztropine or benztropine alone. Data are mean ± SEM of 8 tumor volume under each condition. *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle

Fig. 12

In vitro effects of oxaliplatin associated or not with benztropine on cell viability, GSH and ROS production. Viability was expressed as percent ± SEM versus cells in culture medium alone (100% viability) in CT26 cells (a) and N2a cells (b). GSH was measured using the monochlorobimane dye in CT26 cells (c) and N2a cells (d). ROS generation was measured using H2DCFDA fluorescent emission when converted to the highly fluorescent DCF in CT26 cells (e) and N2a cells (f). Data from at least 4 independent experiments have been pooled and were expressed as means ± SEM of triplicates. *p < 0.05, **p < 0.01 versus oxaliplatin