The Role of TLR4 in the Paclitaxel Effects on Neuronal Growth In Vitro

Paclitaxel (Pac) is an antitumor agent that is widely used for treatment of solid cancers. While being effective as a chemotherapeutic agent, Pac in high doses is neurotoxic, specifically targeting sensory innervations. In view of these toxic effects associated with conventional chemotherapy, decreasing the dose of Pac has been recently suggested as an alternative approach, which might limit neurotoxicity and immunosuppression. However, it remains unclear if low doses of Pac retain its neurotoxic properties or might exhibit unusual effects on neuronal cells. The goal of this study was to analyze the concentration-dependent effect of Pac on isolated and cultured DRG neuronal cells from wild-type and TLR4 knockout mice. Three different morphological parameters were analyzed: the number of neurons which developed neurites, the number of neurites per cell and the total length of neurites per cell. Our data demonstrate that low concentrations of Pac (0.1 nM and 0.5 nM) do not influence the neuronal growth in cultures in both wild type and TLR4 knockout mice. Higher concentrations of Pac (1-100 nM) had a significant effect on DRG neurons from wild type mice, affecting the number of neurons which developed neurites, number of neurites per cell, and the length of neurites. In DRG from TLR4 knockout mice high concentrations of Pac showed a similar effect on the number of neurons which developed neurites and the length of neurites. At the same time, the number of neurites per cell, indicating the process of growth cone initiation, was not affected by high concentrations of Pac. Thus, our data showed that Pac in high concentrations has a significant damaging effect on axonal growth and that this effect is partially mediated through TLR4 pathways. Low doses of Pac are devoid of neuronal toxicity and thus can be safely used in a chemomodulation mode. © 2013 Ustinova et al

Effect of TLR4 inhibitor LPS-RS on the morphological parameters of control, paclitaxel-treated and LPS-treated neurons.

<p>DRG cultures were prepared from wild type animals as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056886#s2" target="_blank">Materials and Methods</a>. Pac was added to cultures at 100 nM; LPS at concentration of 0.5 µg/ml and LPS-RS at concentration of 5 µg/ml. A, percent of neurons with neurites; B, number of neurites per cell; C, total length of neurites per cell. *, p<0.05 versus no LPS-RS; #, p<0.05 versus respective control group (N = 5, one-way ANOVA).</p

Effects of paclitaxel on the number of neurites per cell in control and treated DRG cultures.

<p>DRG cultures were prepared from wild type and TLR4 knockout mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056886#s2" target="_blank">Materials and Methods</a>. Pac was added to cultures at concentrations 0.1 nM–100 nM. Pac inhibits neurite development in a concentration-dependent manner in wild type mice (solid bars), but not in TLR4 knockout mice (open bars). *, p<0.05 versus 0 M of Pac in the same group; #, p<0.05 vs. wild type at the same concentration of Pac (N = 5, one-way ANOVA).</p

Effects of paclitaxel on the total length of neurites per cell in control and treated DRG cultures.

<p>DRG cultures were prepared from wild type and TLR4 knockout animals as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056886#s2" target="_blank">Materials and Methods</a>. Pac was added to cultures at concentrations 0.1 nM–100 nM. As shown, Pac decreases the length of neurites in a concentration-dependent manner both in control (solid bars) and TLR4 knockout mice (open bars). *, p<0.05 versus 0 M of Pac in the same group; #, p<0.05 versus wild type at the same concentration of Pac. (N = 5, one-way ANOVA).</p

Cell morphology of neurons in DRG cultures (high power view).

<p><b>A</b>. In control cultures, neurons develop numerous well defined neurites with multiple extensive branching after 48 h of incubation. <b>B</b>. Neurons incubated with high concentrations of Pac (100 nM) develop multiple short processes, but no well-formed neurites. Representative pictures are shown from >5 independent experiments with similar results.</p

Effects of paclitaxel on the number of neurons with neurites in control and treated DRG cultures.

<p>DRG cultures were prepared from wild type and TLR4 knockout animals as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056886#s2" target="_blank">Materials and Methods</a>. Pac was added to cultures at concentrations 0.1 nM–100 nM. As shown, Pac reduces the number of regenerating neurons in a concentration-dependent manner, both in DRG cultures obtained from control wild type (solid bars) and TLR4 knockout mice (open bars). *, p<0.05 versus 0 M of Pac in the same group (N = 5, one-way ANOVA).</p