Anticonvulsant effects of novel and repurposed drugs on docetaxel-induced neuropathy in C. elegans

Paola Ximena Gonzalez-Lerma; Crystal Lloyd; Scarlet J Park; Ken Dawson-Scully

doi:10.1371/journal.pone.0342236

. 2026 Feb 6;21(2):e0342236. doi: 10.1371/journal.pone.0342236

Anticonvulsant effects of novel and repurposed drugs on docetaxel-induced neuropathy in C. elegans

Paola Ximena Gonzalez-Lerma ^1,^2,^3,^¤a, Crystal Lloyd ^3,⁴, Scarlet J Park ^3,^¤b, Ken Dawson-Scully ^1,^3,^5,^¤c,^*

Editor: Michael Massiah⁶

PMCID: PMC12880658 PMID: 41650182

Abstract

Chemotherapeutic agents used for most common cancers are frequently associated with neurotoxicity, which often include debilitating side effects such as seizures. Docetaxel, one of the most widely and effectively used chemotherapeutic drugs, is associated with an array of symptoms referred to as Docetaxel-Induced Peripheral Neuropathies (DIPNs), including motor neuropathy, tingling, muscle weakness, and numbness. In this study, we use the electroconvulsive assay to model DIPN-related muscle weakness in C. elegans, via shock induction. We show that acutely or chronically exposing nematodes to docetaxel increases time to recovery from shock-induced seizure-like behaviors. Additionally, we find that sildenafil citrate, a PDE-5 inhibitor, and a novel bicyclic bridge compound, Resveramorph-3 (RVM-3), are both effective at rescuing the animals from prolonged seizure-like movement duration following acute and chronic exposure to docetaxel. The results demonstrate that sildenafil citrate and RVM-3 are potential candidates for mitigating the neurological deficits resulting from DIPNs.

Introduction

Although cancer remains a leading cause of death globally, significant strides in its diagnosis and treatment have dramatically increased survival rates. For example, 69% of cancer patients in the U.S. receive a 5 + year survival prognosis [1]. Consequently, addressing the long-term toxicity of cancer treatments is increasingly critical for quality of life [2].

Chemotherapy treatments, while effective against cancer, often result in significant long-term toxicities. Among these, Chemotherapy-Induced Peripheral Neuropathies (CIPNs) are particularly debilitating, affecting the central and peripheral nervous systems and impacting up to 85% of the patients and survivors of cancer [3]. Docetaxel is a taxane which disrupts microtubule-mediated cell division and the dynamic assembly of polymer microtubule subunits, triggering peripheral neuropathies through damaged nerve terminals, neuronal axons, and cellular mitochondria [4]. Intravenously administered docetaxel has been shown to be effective for treating advanced, metastatic, or chemotherapy-resistant cancers, such as androgen-independent prostate cancer and head and neck squamous carcinoma, but its neurotoxic side effects often lead patients to abandon treatment [5,6].

Studies have shown that several drugs used in the treatment of cancer, can trigger seizures [7]. Furthermore, there have been reported cases of docetaxel infusion leading to dose-dependent peripheral sensory [8] or motor [9] neuropathy in cancer patients. Exposure to taxanes, which includes docetaxel, can result in reduced motor and sensory nerve action potentials, along with decreased motor nerve conduction velocity [10–12]. Other studies in breast cancer patients treated with docetaxel reported motor peripheral neuropathy as more common than sensory peripheral neuropathy [13]. In this work we focus on the effects that docetaxel has in modulating time to recovery from shock-induced seizure-like behaviors in treated Caenorhabditis elegans.

The genetically tractable nematodes have a mapped neuronal circuitry, which facilitates identification of neuronal mechanisms that correlate with locomotive phenotypes [14–16]. Previous works have established C. elegans as a useful model for studying the cellular and molecular basis for structural changes in axons [17] that accompany peripheral neuropathy, particularly including following exposure to paclitaxel, a taxane [17]. As taxanes mainly disrupt microtubule-mediated transport and axonal integrity, we hypothesized that docetaxel, which is another taxane, may induce similar mechano-toxic effects observed following paclitaxel treatment in nematodes.

Our lab has previously used electroshock to model seizure-like behaviors in C. elegans, including drug exposure [18–21]. In this study, we use C. elegans to establish a model of shock-induced seizure-like behaviors and evaluate drug-induced changes in recovery time for the purpose of identifying potential rescue agents. We show that acute and chronic exposure to docetaxel increases the duration and severity of shock-induced seizure-like behaviors in worms. Furthermore, these docetaxel-induced effects were alleviated by concurrent administration of sildenafil citrate, an indirect protein kinase G (PKG) activator [20], and Resveramorph-3, a novel bicyclic compound [22]. The results from this study identify potential candidates for mitigating the neurological deficits resulting from DIPNs.

Materials and methods

C. elegans stocks and cultivation

All animals used in this study were C. elegans of the N2 strain, a commonly used control strain. The worms were acquired from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota. Stocks were maintained and transferred every 3–4 days on standard Nematode Growth Medium (NGM) agar plates seeded with OP50 E. coli. The animals were reared and maintained in a temperature-controlled setting (20 °C, except the day before the experiment). Worms were transferred using a platinum wire pick, sterilized between transfers by a butane flame.

Acute exposure protocol

On day 1, adult worms with eggs were plated on NGM agar plates seeded with OP50 E. coli for 2 days at 20 °C. On day 3, L4-stage worms were picked and plated on a new NGM agar plate seeded with OP50 E. coli for overnight incubation at 22 °C. On day 4, 1-day-old adult worms were incubated for 30 minutes in M9 saline (control) or in the respective test solution prior to the electroshock delivery. Docetaxel and sildenafil citrate were first dissolved in 1% dimethyl sulfoxide (DMSO), followed by a 1:10 dilution in M9 saline. Therefore, the tested solutions were dissolved in 0.1% DMSO of the total volume with a 99.9% M9 saline. RVM-3 was dissolved directly in M9 saline.

Chronic exposure protocol

For chronic exposure, E. coli-seeded NGM agar plates were coated with M9 saline containing the specified concentration of docetaxel or vehicle control (M9 with 0.1% v/v DMSO; see next section). In detail, docetaxel was first dissolved in 1% DMSO, followed by a 1:10 dilution in M9 saline (this is equivalent to the acute experiments). Therefore, the tested solutions were dissolved in 0.1% DMSO of the total volume with a 99.9% M9 saline. As for plate preparation, 10 milliliters of NGM agar were first dispensed per plate and once the agar solidified, 2 milliliters of corresponding solutions were poured on the surface of the agar plates. Therefore, for chronic concentrations, DMSO was further dissolved in a total of 12 milliliters, resulting in a final DMSO concentration of ~0.01%, rather than the 0.1% used for acute experiments. The final drug concentrations used for chronic experiments take into account the additional volumes. Plates were then covered and left to dry for 24 hours, away from light at room temperature, to avoid light and temperature degradation. All plates used in this protocol were standardized. After plates air-dried with the coated layer of corresponding solution, these were ready to use for experiments. Ten plates were prepared per condition. New batches of plates were prepared every week to avoid drug degradation through time. Using the electroshock assay protocol, we tested each chronic plate per condition against the acute protocol to ensure compounds distributed evenly and retained bioactivity on the agar. For experiments, nematodes were placed on the surface of each plate. It is important to note that C. elegans do not burrow into the agar, but rather swim on the surface of the agar. On day 1, six gravid adult worms were transferred onto the surface of each coated plate and incubated for 2 days at 20 °C. On day 3, L4-stage worms were transferred to a new drug-treated plate for overnight incubation at 22 °C. On day 4, 1-day-old adult worms were incubated for 30 minutes in M9 saline (control) or the test solution, prior to the electroshock delivery.

Drugs and chemicals used

Drugs and solutions used in the electroconvulsive shock assay were: M9 saline (0.022M KH₂PO₄, 0.042M Na₂HPO₄, 0.085M NaCl, 0.008M MgSO₄), docetaxel (ThermoFisher Scientific, CAS: 114977-26-5), and sildenafil citrate (Viagra®; Alabama Pharmacy Industry Solutions, CAS: 171599-83-0). Resveramorph-3 (RVM-3) was synthesized by the Lepore Lab at Florida Atlantic University. Docetaxel and sildenafil citrate were dissolved first in 1% DMSO and subsequently diluted 1:10 in M9 saline to achieve the final desired drug concentrations. This means that tested solutions contained 0.1% DMSO and 99.9% M9 saline. DMSO alone could have neuroprotective properties when employed at low concentrations. However, previously in our lab a DMSO dose curve was completed showing that there is no significant difference in recovery from electric shock in concentrations up to 0.5% DMSO [19]. RVM-3 is a novel compound previously synthesized and studied for solubility by our lab [22]. RVM-3 has a molecular weight of 418.44 g/mol. For this study, lyophilized RVM-3 was stored in parafilm sealed amber glass vials away from light at 20 °C for a period of up to three months. The day of experiments, 1 milligram of lyophilized RVM-3 was resuspended directly into M9 saline to make a stock concentration of 100 µM. RVM-3 solutions were stored in parafilm sealed amber glass vials away from light at 20 °C for a period of up to two weeks.

Electroshock assay

C. elegans possess 302 neurons, conserved neurotransmitter systems, a simplified neuronal circuitry, and a fully mapped connectome, making it a powerful model for neurological studies. Building on our previous work [18–21], we established an electroshock assay for pharmacological screening. This assay elicits a shock-induced seizure-like behavior that serves as a behavioral indicator of locomotor circuitry and neuronal function, enabling the identification of compounds that influence recovery time from these behaviors. The electroshock assay was conducted as previously published [18–21] with minor modifications. For the electroconvulsive shock assay, the setup consisted of a Grass SD9 stimulator, Grass SD44 stimulator, a dissecting stereoscope (AmScope SM-1TSX) with a camera (HY-1139), and a computer with OBS Studio recording software. Briefly, approximately six 1-day-old adult C. elegans were transferred into silicone tubes. Following 30 minutes of incubation, two 18-gauge copper wires were inserted into either end of the tube to a 1-cm distance between the electrodes. Alligator clips were attached to each electrode and connected to the stimulator. Worms were monitored for 30 seconds before and 5 minutes after a 3-sec shock delivery (200 Hz, 47 V). New electrodes were used for each test solution, and the experimental tubes were discarded after each trial. The shock delivery was visually confirmed as electrolysis in the form of bubbles released from either electrode. Video recordings were analyzed for the recovery time of each worm after the shock-induced seizure-like behaviors. Nematodes occluded from view or those that did not display normal movement before and after shock were excluded from analysis. Animals that recovered within the five-minute window of recording were counted as recovered. Recovery time of a worm was defined as time at which the nematode initiated three consecutive sinusoidal wave-like swimming motions, without considering the speed or depth of the wave. Animals that were active prior to shock but did not recover following shock, were considered as non-recovered (NR) and were used to calculate percent non-recovery (%NR) for each solution tested using the following formula: $% NR = \frac{(Number of non - recovered worms)}{(Total number of worms analyzed)} \times 100$ . For accurate screening, we ran six tubes per condition, each tube containing six nematodes. To avoid day effects on our screening, we ran two tubes per condition per day. For clarification, each day we included a trial of M9 saline as a negative control. To maintain drug screening and video analysis objectivity, two researchers were involved in collecting data for this study. Each researcher was responsible for completing randomly assigned dosage curves on different days of the week as mentioned previously. To eliminate personal bias during the video analysis process, videos were swapped between the two researchers and scores were blinded to condition.

Statistical methods

For recovery time, one-way ANOVA was followed by all pairwise Student-Newman-Keuls post hoc test. Chi-square tests were used to compare % NR. Detailed statistical results can be found in the supplemental material. An alpha value of 0.05 to determine significance. All ANOVA statistics were performed using SigmaPlot (Version 13) and Chi-Square Tests were performed using the Standard Deviation Chi-Square Calculator.

Results

We first tested whether docetaxel is an agent that can increase time to recovery from shock-induced seizure-like behaviors in C. elegans. Acute exposure to 0.005, 0.01, and 1 mM docetaxel significantly increased time to recovery [Fig 1A] and percent non-recovery (%NR) [Fig 1B] following electroshock, as compared to M9 saline.

Fig 1 — **(A)** Acute exposure to docetaxel increases time to recovery in a concentration-dependent manner. Different letters denote a statistically significant difference in the mean values between the groups, where “a” stands for not statistically significantly different from M9 saline and “b” stands for statistically significantly different from M9 saline (Student-Newman Keuls, p < 0.05). Data shown as mean ± s.e.m. **(B)** Acute exposure to increasing concentrations of docetaxel increases the percentage of non-recovered worms following the electroshock. 0.001 mM DTX vs. M9, X²= 16.6071, p < 0.0001; 0.002 mM DTX vs. M9, X²= 18.7615, p < 0.0001; 0.0035 mM DTX vs. M9, X²= 19.3063, p < 0.0001; 0.005 mM DTX vs. M9, X²= 21.698, p < 0.0001; 0.01 mM DTX vs. M9, X²= 20.7299, p < 0.0001; 1 mM DTX vs. M9, X²= 24.2858, p < 0.0001. N > 30 for each group. ****, *p <* 0.0001, compared to M9.

To simulate seizure-like behavior severity observed in cancer patients undergoing prolonged chemotherapeutic treatments, we chronically exposed the nematodes to varying concentrations of docetaxel. Chronic exposure to docetaxel increased time to recovery from shock-induced seizure-like behaviors [Fig 2A] and % NR [Fig 2B], in contrast to nematodes exposed to M9 saline, which did not exhibit such changes.

Fig 2 — **(A)** Chronic exposure to docetaxel increases time to recovery with increasing concentrations. Different letters denote a statistically significant difference in the mean values between the groups where “a” stands for not statistically significantly different from M9 saline, “b” stands for statistically significantly different from M9 saline, and “c” stands for statistically significantly different from M9 saline and solutions labeled “b” (Student-Newman Keuls, p < 0.05). Data shown as mean ± s.e.m. **(B)** Chronic exposure to increasing concentrations of docetaxel increases the percentage of non-recovered worms following the electroshock. C.E. 0.0035 mM DTX vs. M9, X²= 11.9656, *p =* 0.0005; C.E. 0.005 mM DTX vs. M9, X²= 8.4176, p = 0.0037; C.E. 1 mM DTX vs. M9, X²= 24.7284, p < 0.0001. N > 30 for each group. The horizontal reference line (0.01 mM Docetaxel Acute Exposure) indicates the mean value observed for acute exposure to 0.01 mM docetaxel [Fig 1]. **, *p <* 0.01; ***, *p <* 0.001; ****, *p <* 0.0001; all compared to M9.

Sildenafil citrate (SC), the generic formulation of Viagra®, indirectly activates potassium channel conductance [23]. Acute treatment with 0.5 mM SC had no effect on time to recovery [Fig 3A]. However, when combined with acute exposure to 0.01 mM docetaxel, SC significantly suppressed the time to recovery at concentrations as low as 0.06 mM, although the protective effect was not statistically significant at the two highest concentrations of SC [Fig 3A]. Acute exposure to 0.5 mM SC on its own increased % NR over M9 saline [Fig 3B]. Additionally, co-administration of SC, at concentrations as low as 0.06 mM, suppressed the increased % NR following acute 0.01 mM docetaxel [Fig 3B].

Fig 3 — **(A)** Acute treatment with sildenafil citrate significantly decreases time to recovery for worms acutely treated with 0.01 mM docetaxel. Different letters denote a statistically significant difference in the mean values between the groups where “a” stands for not statistically significantly different from M9 saline, “b” stands for statistically significantly different from M9 saline, and “a/b” stands for not statistically significantly different from M9 saline or solutions labeled “b” (Student-Newman Keuls, p < 0.05). Data shown as mean ± s.e.m. **(B)** Acute treatment with sildenafil citrate decreases the percentage of non-recovered worms following the electroshock. 0.5 mM S.C. vs. M9, X²= 2.9668, p = 0.0850; 0.01 mM DTX vs. M9, X²= 25.0325, p < 0.0001; 0.01 mM DTX. vs. 0.06 mM S.C. + 0.01 mM DTX, X²= 28.1521, p < 0.0001; 0.01 mM DTX vs. 0.125 mM S.C. + 0.01 mM DTX, X²= 23. 4833, p < 0.0001; 0.01 mM DTX vs. 0.25 mM S.C. + 0.01mM DTX, X²= 24.9289, p < 0.0001; 0.01 mM DTX vs. 0.5 mM S.C. + 0.01 mM DTX, X²= 29.0098, *p <* 0.0001; 0.01 mM DTX vs. 1 mM S.C. + 0.01 mM DTX, X²= 18.9077, p < 0.0001. N > 30 for each group. ****, *p <* 0.0001, compared to M9. *####, p <* 0.0001, compared to 0.01 mM DTX. S.C., Sildenafil Citrate; DTX, Docetaxel.

Resveramorph-3 (RVM-3) is a member of a family of bridged bicyclic compounds inspired by resveratrol [22]. Based on its structure and similarity to related compounds, RVM-3 has been proposed to function as an irreversible agonist, although its mechanism of action has not been established [24]. In our experiments, 100 µM RVM-3, when combined with acute exposure to 0.01 and 1 mM docetaxel, significantly reduced shock-induced seizure-like behavior duration [Fig 4A] and % NR [Fig 4B].

Fig 4 — **(A)** Acute treatment with RVM-3 significantly decreased time to recovery for worms treated with 0.01 and 1 mM docetaxel. Different letters denote a statistically significant difference in the mean values between the groups where “a” stands for not statistically significantly different from M9 saline, and “b” stands for statistically significantly different from M9 saline (Student-Newman Keuls, p < 0.05). Data shown as mean ± s.e.m. **(B)** Acute treatment with 100 µM RVM-3 significantly decreases the percentage of non-recovered worms treated with 1 mM docetaxel. 100 µM RVM3 vs. M9, X²= 0.3233, p = 0.5696; 0.01 mM DTX vs. M9, X²= 13.1767, p < 0.0001; 1 mM DTX vs. M9, X²= 38.3749, p < 0.0001; 0.01 mM DTX vs. 0.01 mM DTX + 100 µM RVM3, X²= 1.5921, p = 0.2070; 1 mM DTX vs. 1 mM DTX + 100 µM RVM3, X²= 12.2982, *p <* 0.0005. N > 30 for each group. ***, *p <* 0.001; ****, *p <* 0.0001; all compared to M9. *###, p <* 0.001, compared to 1 mM DTX. RVM-3, Resveramorph 3.

When combined with chronic exposure to 0.005 mM docetaxel, 0.1 and 0.25 mM acute SC suppressed time to recovery [Fig 5A], and 0.25 mM SC suppressed %NR below control levels [Fig 5B]. Acute treatment with 100 µM RVM-3 significantly decreased time to recovery when the animals were chronically exposed to 0.005 mM docetaxel [Fig 6A]. However, RVM-3 did not suppress the increased % NR with chronic 0.005 mM docetaxel exposure [Fig 6B]. Additionally, acute treatment with 100 µM RVM-3 significantly decreased the time to recovery [Fig 7A] and decreased % NR [Fig 7B] in animals subjected to a chronic treatment of 3.5 or 10 μM docetaxel.

Fig 6 — **(A)** Acute treatment with RVM 3 significantly decreases time to recovery for worms treated with chronic 0.005 mM docetaxel. Different letters denote a statistically significant difference in the mean values between the groups where “a” stands for not statistically significantly different from M9 saline, and “b” stands for statistically significantly different from M9 saline (Student-Newman Keuls, P < 0.05). Data shown as mean ± s.e.m. **(B)** Acute treatment with RVM 3 does not decrease the percentage of non-recovered worms subjected to chronic 0.005 mM docetaxel exposure. 100 µM RVM3 vs. M9, X²= 0.1079, p = 0.7425; C.E. 0.005 mM DTX vs. M9, X²= 3.6308, p = 0.0567; C.E. 0.005mM DTX vs. 100 µM RVM3 + C.E. 0.005 mM DTX, X²= 0.0021, p = 0.9634. *, *p <* 0.05, compared to M9. RVM-3, Resveramorph 3; C.E DTX, chronic exposure to 0.005 mM docetaxel.

Fig 7 — **(A)** Acute treatment with RVM-3 significantly decreased time to recovery in worms treated with acute and/or chronic 0.0035 and 0.01 mM docetaxel. Different letters denote a statistically significant difference in the mean values between the groups where “a” stands for not statistically significantly different from M9 saline, “b” stands for statistically significantly different from M9 saline, “c” stands for statistically significantly different from M9 saline and other labeled solutions, and “d” stands for statistically significantly different form M9 saline and other labeled solutions (Student-Newman Keuls, P < 0.05). Data shown as mean ± s.e.m. **(B)** Acute treatment with RVM-3 decreased %NR in animals treated with chronic 0.0035 and 0.01 mM docetaxel, following the electroshock. 100 µM RVM3 vs. M9, X²= 0.0888, p = 0.7657; Acute 0.0035 mM DTX vs. M9, X²= 12.1153, *p <* 0.0001; C.E. 0.0035 mM DTX in M9 vs. M9, X²= 9.7686, p = 0.0018; C.E. 0.0035 mM DTX in 0.0035 mM DTX vs. M9, X²= 9.1983, p = 0.0024; C.E. 0.0035 mM DTX in 100 µM RVM3 vs. M9, X²= 0.5822, p = 0.4455; Acute 0.01 mM DTX vs. M9, X²= 10.4772, p = 0.0012; C.E. 0.01 mM DTX in M9 vs. M9, X²= 10.3204, p = 0.0013; C.E. 0.01 mM DTX in 0.01 mM DTX vs. M9, X²= 12.8355, *p <* 0.0001; C.E. 0.01 mM DTX in 100 µM RVM3 vs. M9, X²= 1.2115, p = 0.2710; C.E. 0.01 mM DTX in 0.01 mM DTX + 100 µM RVM3 vs. M9, X²= 1.1258, p = 0.2887. N > 30 for each group. **, *p <* 0.01; ***, *p <* 0.001; ****, *p <* 0.0001; all compared to M9. RVM-3, Resveramorph 3; DTX, Docetaxel.

Discussion

Docetaxel kills cancerous cells by disrupting the dynamic assembly of polymer microtubule subunits, which leads to Docetaxel-Induced Peripheral Neuropathies (DIPNs) [25]. In severe cases, DIPN-induced muscle weakness often leads patients to abandon their chemotherapy regimen, and there is an urgent need for effective agents and/or therapies that mitigate and/or prevent the development of DIPNs. In this study, we acutely or chronically exposed C. elegans to docetaxel, demonstrating that the drug can modulate time to recovery from shock-induced seizure-like behaviors in the invertebrate model. Based on our findings we can hypothesize that nematodes treated with acute docetaxel exposure displayed an increase in time to recovery from shock-induced seizure-like behaviors [Fig 1A]. We attribute drug effects seen from acute exposure to be concentration dependent such that after reaching threshold, drug toxicity led to the display of increased time to recovery from shock-induced seizure-like behaviors in nematodes. Regarding nematodes treated with chronic docetaxel exposure, we observed increased time to recovery from shock-induced seizure-like behaviors, unlike nematodes exposed to M9 saline, which did not exhibit such behaviors [Fig 2A]. In this scenario, we attribute drug effects to be due to time dependent toxicity with sensitization, not purely concentration dependent effects.

The NO-cGMP/PKG pathway is a potential target for seizure therapeutics, as it influences downstream K⁺ channel conductance through key players like PKG and PP2A [23]. Additionally, repurposing of sildenafil citrate has gained significant interest within the pharmaceutical and healthcare communities, as it shows potential to improve drug tolerability and efficacy of chemotherapy [26]. In this study, we show that acute treatment with sildenafil citrate, a PKG pathway activator [23], significantly reduces time to recovery from shock-induced seizure-like behaviors following acute [Fig 3A] and chronic [Fig 5A] exposure to docetaxel.

Docetaxel inhibits K⁺ currents in a dose-dependent manner [27], whereas sildenafil citrate activates the NO/cGMP/PKG pathway, possibly promoting K⁺ conductance as speculated by previous work in our lab [23]. Furthermore, the effects of sildenafil have been linked to the GABAergic system [28], suggesting that sildenafil citrate reduces neuronal excitability through mechanisms involving both K⁺ channels and GABA signaling, ameliorating DIPN-related effects. Being a PDE5-inhibitor, sildenafil works as a vasodilator, following the increase in cyclic guanosine (cGMP) levels, leading to smooth muscle relaxation and vasodilation [28] which can promote neuroprotective effects. In this study, sildenafil citrate significantly reduced recovery time following shock-induced seizure-like behaviors [Fig 3, 5], consistent with prior findings that sildenafil citrate decreases recovery time in this assay [29]. Based on these observations, we interpret sildenafil citrate as a compound that reduces recovery time in this behavioral paradigm without evidence of altering shock induction. In terms of toxicity, from our findings [Fig 3, 5] we observed that acute co-exposure to high-dose sildenafil citrate and 0.01 mM docetaxel increased seizure-like behaviors, whereas chronic docetaxel exposure followed by acute sildenafil citrate treatment did not produce this effect. This pattern suggests a time-dependent interaction in which simultaneous exposure may transiently heighten sensitivity to seizure-like behaviors, while chronic docetaxel exposure may induce adaptive responses that mitigate this sensitivity. This underlying mechanism remains unclear and warrants further investigation.

Moreover, resveratrol-inspired compounds, known as Resveramorphs (RVM), possess a three-dimensional structure that increases complementarity to a binding site leading to improved target selectivity and suggesting potential neuroprotective effects [22,24]. Our lab has previously demonstrated that in C. elegans, shock-induced seizure-like behaviors decreased in duration when worms were exposed to acute combinations of Resveramorph-3 (RVM-3) and pentylenetetrazol (PTZ) solutions [24]. In our study, acute exposure to 100 µM RVM-3 in combination with docetaxel decreased the duration of shock-induced seizure-like behaviors [Fig 4]. Nematodes grown chronically in docetaxel and then treated acutely with RVM-3 showed a similar reduction [Fig 6]. These findings suggest that RVM-3 can reduce recovery time from seizure-like behaviors under both acute and chronic conditions. One interpretation is that the pathways or processes influenced by RVM-3 remain responsive regardless of prior docetaxel exposure; however, the basis of these effects is not yet understood. RVM-3 is a novel compound and therefore further studies are required to evaluate its potential toxicity, define dose-dependent effects, and determine whether the observed reductions in recovery time reflect specific actions within this behavioral paradigm or broader effects on neuronal or locomotor function.

The mechanism by which docetaxel modulates time to recovery from shock-induced seizure-like behaviors in C. elegans is not clearly defined in this study, as electrophysiological changes during drug exposure were not examined. While there is no direct evidence linking docetaxel to axonal or neuronal degeneration in nematodes, prior studies show taxol-induced mechanotoxicity [17], therefore, we speculate the possibility of similar axonal damage from docetaxel. Docetaxel-induced neuropathy is typically mild to moderate, but high cumulative doses can cause severe clinical and electrophysiological abnormalities, with nerve biopsies in humans showing large, myelinated fiber loss and occasional axonal degeneration [3]. Future studies could test the effects of PKG activators on other proconvulsant such as PTZ or glyphosate-based compounds [18].

Future work may validate the involvement of the NO/cGMP/PKG pathway by using a secondary PDE5 inhibitor, such as tadalafil, or using genetic mutants of the PKG homolog egl-4 [29], or of relevant cGMP phosphodiesterases (pde-1, pde-5) [23]. Additionally, replicating the anticonvulsant properties of sildenafil citrate and RVM-3 in mammalian models of seizure-like behaviors will prove useful for assessing their potential as a combinatorial therapeutic. Inclusion of full dose-response model fitting curves and associated EC50/IC50 values would provide additional quantitative insight. However, the primary aim of this study is to establish and demonstrate the utility of our electroshock assay as a screening platform rather than to generate complete pharmacological profiles for each compound. In this study, the concentration-dependent assessments are qualitative and exploratory, therefore, we acknowledge that there is a need for future studies to perform a full-dose response characterization.

Supporting information

S1 Fig. One-way ANOVA: Acute docetaxel treatment increases time to recovery from shock-induced seizure-like behaviors with increasing concentration in C. elegans.

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S2 Fig. Chi-square test: Acute docetaxel treatment increases time to recovery from shock-induced seizure-like behaviors with increasing concentration in C. elegans.

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S3 Fig. One-way ANOVA: Nematodes treated with chronic docetaxel, display an increase in time to recovery from shock-induced seizure-like behaviors when compared to nematodes exposed to M9 saline alone.

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S4 Fig. Chi-square test: Nematodes treated with chronic docetaxel, display an increase in time to recovery from shock-induced seizure-like behaviors when compared to nematodes exposed to M9 saline alone.

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S5 Fig. One-way ANOVA: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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S6 Fig. Chi-square test: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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S7 Fig. One-way ANOVA: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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S8 Fig. Chi-square test: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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S9 Fig. One-way ANOVA: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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S10 Fig. Chi-square test: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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S11 Fig. One-way ANOVA: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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S12 Fig. Chi-square test: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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S13 Fig. One-way ANOVA: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors associated with acute and chronic exposure to docetaxel.

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S14 Fig. Chi-square test: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors associated with acute and chronic exposure to docetaxel.

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pone.0342236.s014.tif^{(154.1KB, tif)}

S15 Fig. DMSO effects on C. elegans using the electroshock assay. A DMSO sham concentration curve at 0.1% and 0.5%, demonstrates these concentrations have no significant effect on wild-type recovery time after electric shock.

(TIF)

pone.0342236.s015.tif^{(75.9KB, tif)}

S16 Fig. RVM-3 concentration recovery comparisons. Recovery time for N2 worms in M9 saline or in the presence of PTZ.

(TIF)

pone.0342236.s016.tif^{(145KB, tif)}

Acknowledgments

We thank members of the Dawson-Scully Lab for feedback on the writing of the manuscript. C. elegans strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

Data Availability

All datasets generated and/or analyzed during the current study are available in Open Science Framework DOI: 10.17605/OSF.IO/S56DM.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0342236.r001

Decision Letter 0

Michael Massiah

6 Nov 2025

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Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

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Reviewer #1: Overview

This manuscript investigates the effects of a chemotherapeutic, docetaxel, in an electroconvulsive assay using the model organism C. elegans. Additionally, the authors identify two drugs that ameliorate the docetaxel effects – sildenafil citrate and resveramorph-3.

Overall, the results appear convincing, although there are some concerns about experimental protocols and phrasing that need correcting.

Comments

Acute exposure. What is the final concentration of DMSO for the docetaxel and sildenafil citrate that the worm experiences? It is not clear from the methods whether an equivalent volume of DMSO is used in the controls. DMSO itself can have neuroprotective properties at low concentrations and needs to be included in your controls.

Chronic exposure. With the chronic exposure experiments, however, you are dissolving solutions into your NGM plate. Therefore the chronic drug concentration experienced by worm will by reduced by a factor dependent on the volume of the plate. If your plate has 10 mls of NGM in it, the stated concentrations of docetaxel will be reduced by a factor of 10. And that assumes that the plate volume is standardised (is it?) and that the drug efficacy is not time-dependent (is it?). The authors need to clarify their experimental protocol and what the actual stated concentrations for the chronic experiments were.

Additionally, the wording in Figure 2 implies that chronic treatment “increases severity of behaviours”. But the statistics appear to be done only against chronic treatment of M9. Comparing Figures 1 and 2 – the data look pretty similar, indicating a lack of chronic response. Although given the point above about the chronic concentrations, this experiment may simply be replicating the acute effects. Do the authors have any evidence for an increase in behavioural severity?

Phrasing. The authors do not measure seizures or induction of seizures – they are specifically measuring time-to-recovery or lack of recovery – which is different. In a number of places the authors have misconstrued the experimental outcomes, which needs to be corrected. Specifically:

• Line 140. A proconvulsant is an agent that initiates or lowers the threshold for seizures. The authors should either test for a proconvulsant effect (eg. Identify whether the voltage threshold for seizures is actually lowered) or reword for what is actually shown.

• Line 176 states that “docetaxel models DIPN-seizure behaviour in the worm”. My understanding is that docetaxel is a proconvulsant and there isn’t evidence that it affects time-to-recovery from seizures. This statement needs to be supported or reworded.

• Line 197 specifically states “docetaxel induces seizure-like behaviors in C. elegans”, which is not supported by the results and needs to be reworded.

Minor comments

• Methods line 94. The day after L4-stage worms would be Day 0 Adulthood (not Day 1).

• The rationale for some experiments are not well explained. It isn’t clear to this reader why a neuroprotective effect of Viagra was expected. The authors should justify their choice of putative neuroprotective compounds.

• Figure legends state that “different letters denote a statistically significant difference in the mean values between the Groups” – but then it isn’t explained what “a” and “b” (and later on “a/b”) actually represent. I assume “a” is not significant and “b” is?

• Figure 2. It is unclear why a line for 0.01 mM docetaxel is included when that isn’t being tested chronically.

• Figure 7. It isn’t completely clear here why 3.5 uM is being studied. Nor why it is listed as uM rather than mM (everywhere else in the manuscript).

Reviewer #2: The authors use the C. elegans electroshock paradigm to model docetaxel-associated neurotoxicity and test two anticonvulsant candidates: sildenafil citrate (a PDE-5 inhibitor) and a novel bridged bicyclic compound, Resveramorph-3 (RVM-3). Acute and chronic docetaxel exposure prolong recovery from shock-induced convulsions and increase non-recovery (NR). Sildenafil and RVM-3 reduce seizure-like duration under several conditions. The manuscript is clearly written and addresses a clinically relevant problem (chemotherapy-related neurotoxicity) with a tractable invertebrate model.

Major issues

1. The electroshock assay treats ~6 worms together per tube/shock. Report the number of independent tubes and independent experimental days per condition.

2. Acute test solutions contain 0.1% DMSO (drug dissolved in 1% DMSO then 1:10 in M9), but the stated acute control is M9 saline alone. Please include (and analyze against) an acute 0.1% DMSO vehicle control to match drug conditions; otherwise DMSO effects cannot be excluded.

3. Docetaxel is hydrophobic; “coating NGM plates with M9 containing docetaxel” raises concerns about uniformity and stability. Please specify: volumes applied per plate, drying time, storage/light protection, time from coating to use, and evidence the compound distributes/retains bioactivity on agar. Similarly, RVM-3 is said to be dissolved directly in M9—provide rationale/solubility data, stock concentration, purity, and quality control. These details are critical for reproducibility.

4. The recovery criterion (three sinusoidal waves) is clear, but please state whether scorers were blinded to condition and how worms/tubes were randomized across treatments and days.

5. You repeatedly link seizures to Docetaxel-Induced Peripheral Neuropathies (DIPNs). Seizures are central phenomena; while docetaxel-associated encephalopathy and seizures are reported, seizures are not typically considered a feature of peripheral neuropathy per se. Please reframe as docetaxel-associated neurotoxicity (which can include seizures) rather than “DIPN-related seizures,” unless you provide primary evidence/mechanism tying the peripheral pathology to seizure generation.

6. The Results text states that RVM-3 significantly reduced %NR with both 0.01 mM and 1 mM acute docetaxel, but the figure legend/statistics indicate significance only at 1 mM (0.01 mM + RVM-3 p≈0.21, n.s.).

7. You describe concentration dependence qualitatively. Please include dose–response curves with fitted models where possible (even exploratory), report EC50/IC50 or at least slope parameters, and show per-tube datapoints.

Minor issues

• Mechanistic claims: Tone down language implying mechanism (e.g., “RVM-3 potentially acts as an irreversible agonist by covalently binding…”) unless direct evidence is presented here. Likewise, sildenafil’s effects on K⁺ channels are likely indirect via cGMP/PKG; keep causal language cautious and clearly attributed to prior work.

• Terminology consistency: Use “seizure-like behavior” throughout for C. elegans; avoid unqualified “seizures”. Ensure DTX not “DTC” (typo appears once in a legend) and standardize µM/mM spacing.

• Citations: Consider citing C. elegans cGMP/PKG genetics (e.g., egl-4; endogenous pde genes), to support the proposed pathway testing in future work.

Reviewer #3: The use of C. elegans to screen docetaxel effects is interesting and aids in addressing the effects of this chemotherapeutic agent and means of reducing the effects of seizures and altering the recovery time from a seizure by Resveramorph-3 and sildenafil citrate. One view by researchers, in general, is to show the effects of compounds on as many animal models as possible to examine various potential therapeutic medications as one can learn more about the mechanisms of actions including the potential side effects. This study is done well with very detailed explanations in the methodology. The supplemental data is detailed along with the statistical analysis performed.

There are just a few suggestions for the authors which I fell will help a reader better understand overview of the study and why particular compounds are used.

Minor suggestions:

1. Without having to read papers cited( 18-21 ) can the authors give a little bit of background in this manuscript why electroshock is a good model to induce seizures.

2. I am not sure of the logic to try Sildenafil citrate as compared to more specific seizure reducing medications like levetiracetam or the various other medications. The authors state Sildenafil citrate works in the C. elegans model to reduce the seizure induced by docetaxel. But does Sildenafil citrate reduce the induction of the electroshock itself ? So then the seizure is already dampened to reduce the effect of the electroshock but not necessarily the effect of docetaxel ?

3. In the Discussion it is stated that Docetaxel-Induced Peripheral Neuropathies (DIPNs) while will also cause seizures- related to humans.

So, it is not known if Docetaxel induces seizures in C. elegans or did I miss something ? Or are the seizures only induced by electroshock in this model with ones exposed to Docetaxel?

4. It could strengthen the article by including some discussion about the potential side effects or toxicity of sildenafil citrate and Resveramorph-3 in C. elegans, since understanding any harmful or side effects is important when considering these compounds as treatments.

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PLoS One. 2026 Feb 6;21(2):e0342236. doi: 10.1371/journal.pone.0342236.r002

Author response to Decision Letter 1

16 Dec 2025

Dear Reviewers,

We want to thank the reviewers for their thoughtful and constructive feedback. We have carefully considered each comment and revised the manuscript accordingly. We believe that the added clarifications and details have strengthened the manuscript. Below, we provide our detailed responses to each point.

Reviewer 1:

Major Comments:

Acute exposure

1. What is the final concentration of DMSO for the docetaxel and sildenafil citrate that the worm experiences? It is not clear from the methods whether an equivalent volume of DMSO is used in the controls.

In the acute condition exposures, we diluted docetaxel and sildenafil citrate in 1% dimethyl sulfoxide (DMSO) and subsequently diluted 1:10 in M9 saline. This methodology was also used for chronic condition exposures. This means that for acute condition exposures, the tested solutions contained M9 with 0.1% DMSO, meaning 0.1% of the total volume is DMSO and the remaining 99.9% is M9 saline. We note this detail under “chronic exposure protocol” (Lines 107-111). We also briefly reference this under the section titled “drugs and chemicals used” (Lines 131-136), however we acknowledge we did not clarify this for the acute exposure protocol. We appreciate your attention to detail, as we have included this detailed description under the “acute exposure protocol” section (Lines 102-105).

2. DMSO itself can have neuroprotective properties at low concentrations and needs to be included in your controls.

Thank you for pointing this out. When we implemented DMSO as a dissolving vehicle for our compounds, we also considered the possibility that low concentrations of DMSO could generate neuroprotective effects, therefore, to address this issue, we turned to previously published data by our lab. In previous work, a concentration curve for dimethyl sulfoxide (DMSO) was conducted showing that there is no significant difference in recovery from electric shock in concentrations up to 0.5% DMSO [19]. We have included this dose curve image as a supplemental figure titled S8 Fig. DMSO effects on C. elegans using the electroshock assay. This is important since we accept that DMSO can be used as a solvent for this assay, without affecting the results observed by docetaxel and sildenafil citrate. For further clarification, we make a note of these results in the section titled “drugs and chemicals used” (Lines 131-136). We have also included the figure in this response for your accessibility. Considering that we had previously published work showing that a 0.1% concentration of DMSO did not show significant effects on the recovery-time of wild-type nematodes after electric shock, we did not run extra sets of controls in our work. If you feel like this is something that must be included in our current work, we would be happy to run the extra set of experiments. We like to note that we respectfully value your feedback on this matter and we seek to only justify our original methodology for this part of our study. However, if you do feel this is something that must be repeated we will address it without a problem.

Note: A DMSO sham concentration curve at 0.1% and 0.5%, and demonstrates these concentrations have no significant effect on wild-type recovery time after electric shock [19].

Chronic exposure

3. With the chronic exposure experiments, however, you are dissolving solutions into your NGM plate. Therefore, the chronic drug concentration experienced by worm will by reduced by a factor dependent on the volume of the plate. If your plate has 10 mls of NGM in it, the stated concentrations of docetaxel will be reduced by a factor of 10. And that assumes that the plate volume is standardised (is it?)

For chronic exposure experiments, we coated each plate with M9 saline containing the corresponding concentrations of docetaxel or vehicle control (M9 with 0.1% v/v dimethyl sulfoxide (DMSO). We mention this in the “chronic exposure protocol” section (Lines 107-109). In more detail, 10 milliliters of NGM agar were first dispensed per plate and once the agar solidified, 2 milliliters of corresponding solutions were poured on the surface of the agar plates. Plates were then covered and left to dry for 24 hours, away from light at room temperature, to avoid light and temperature degradation. All plates used in the chronic exposure protocol were standardized. After plates air-dried with the coated layer of corresponding solution, these were ready to use for experiments. New batches of plates were prepared every week to avoid drug degradation through time. For experiments, worms were placed on the surface of the plates. It is important to note that C. elegans do not burrow into the agar, but rather swim on the surface of the agar. A total of 6 gravid adult nematodes were transferred onto the surface of each coated plate and incubated for 2 days at 20 ℃. After 3 days of incubation, L4-stage worms were then transferred to a new drug-coated plate for overnight incubation at 22 ℃. On day 4, 1-day-old adult worms were incubated for 30 minutes in M9 saline (control) or the test solution prior to the electroshock delivery. We have included this detailed description of the protocol under the “chronic exposure protocol” section (Lines 111-125). Regarding acute docetaxel exposure, we observed that drug effects were concentration-dependent such that after reaching a threshold, the drug generated toxicity to the nematode which we observed as an increase in time-to-recovery from the shock-induced seizure-like behaviors (Lines 323-326). In the scenario of chronic drug exposure, we hypothesize that the effects observed were time-dependent with sensitization, not purely concentration effects. This due to the fact that chronic growth could have pre-disposed the nematode to be then affected by the subsequent acute drug exposure, resulting in toxicity seen as an increase in time-to-recovery from shock-induced seizure-like behaviors starting at lower concentrations (Lines 326-329).

4. The authors need to clarify their experimental protocol and what the actual stated concentrations for the chronic experiments were

We have included a detailed description of the chronic exposure protocol in the above response for Question 3. However, to further clarify, we have poured 2 milliliters of the prepared solutions onto the surface of each solidified agar plate. Plates were then covered and left to air-dry for 24 hours, away from light at room temperature to avoid light and temperature degradation. All plates used in the chronic exposure protocol were standardized. After plates air-dried with the coated layer of corresponding solution, these were ready to use for experiments. Additionally, for drug concentration specification, docetaxel was first dissolved in 1% DMSO, followed by a 1:10 dilution in M9 saline. Therefore, the tested solutions were dissolved in 0.1% DMSO of the total volume with a 99.9% M9 saline. This information was added under the “chronic exposure protocol” section (Lines 107-111).

5. Wording in Figure 2 implies that chronic treatment “increases severity of behaviours”. But the statistics appear to be done only against chronic treatment of M9. Comparing Figures 1 and 2 – the data look pretty similar, indicating a lack of chronic response. Although given the point above about the chronic concentrations, this experiment may simply be replicating the acute effects. Do the authors have any evidence for an increase in behavioural severity?

Figure 2 titled “Chronic docetaxel treatment increases severity of seizure-like behaviors”, is meant to show that nematodes exposed to a chronic docetaxel protocol, tend to display a concentration-dependent increase in time-to-recovery from a seizure-like behavior at lower concentrations (Figure 2), than those nematodes exposed to an acute docetaxel protocol (Figure 1). In figure 1, nematodes exposed to 0.0035 mM acute docetaxel experienced a time-to-recovery from seizure-like behaviors similar to worms exposed to M9 saline. However, nematodes exposed to chronic 0.0035 mM docetaxel experienced an increase in time-to-recovery from seizure-like behaviors that was significantly different from M9 saline. In our figures, we display statistical differences by using different letters. In figure 1, M9 saline and acute 0.0035 mM docetaxel were labeled “a”, indicating that both solutions do not display any significantly difference in time-to-recovery from seizure-like behaviors. In figure 2, M9 saline was labeled “a”, whereas chronic 0.0035 mM docetaxel was labeled “b” indicating a statistically significant difference in time-to-recovery from seizure-like behaviors between the two solutions.

To ensure clarity of the data displayed, we are changing the title for figure 2 to “Nematodes treated with chronic docetaxel, displayed an increase in time-to-recovery from seizure-like behaviors starting at lower concentrations” (see Fig 2 in figure files and manuscript Lines 204-205). In figure 2, we also compared the chronic exposure data to the acute exposure data. In this figure, we show that nematodes in M9 saline, column labeled “a”, displayed a statistically significant decreased time-to-recovery from seizure-like behaviors versus worms treated with chronic 0.0035 mM docetaxel labeled “b”. Furthermore, nematodes treated with chronic 0.005 mM and 1 mM docetaxel displayed a statistically significant increase in time-to-recovery different than animals exposed to chronic 0.0035 mM docetaxel and M9, therefore these columns were labeled “c”. As a form of comparison, we included a horizontal solid line in figure 2 to show that on average, nematodes exposed to acute 0.01 mM docetaxel displayed lower time-to-recovery from seizure-like behaviors than those animals exposed to chronic docetaxel concentrations. We indicate that different letters denote statistically significant difference in the mean values between the groups, and we also explain the meaning of each letter in each figure legend (Lines 190-192, 207-210, 229-231, 251-252, 272-274, 288-289, 299-302).

6. Phrasing. The authors do not measure seizures or induction of seizures – they are specifically measuring time-to-recovery or lack of recovery – which is different. In a number of places the authors have misconstrued the experimental outcomes, which needs to be corrected. Specifically:

We appreciate the reviewer for pointing this out. We agree that by mentioning the increase or decrease of seizure-like behaviors, we are conveying the incorrect idea. In this study, we are measuring time-to-recovery from a shock-induced seizure-like behavior, using our previously established electroshock assay [18-21]. Here, shock induces the seizure-like behavior, while exposure to M9 saline versus acute or chronic docetaxel affects the duration of time-to-recovery from the induced seizure-like behavior. Additionally, we are using sildenafil citrate and resveramorph-3 (RVM-3) to measure the effects these have in decreasing the duration of time-to-recovery from the shock-induced seizure-like behaviors once altered by exposure to docetaxel (Lines 39-42). To clarify our methodology and results, we have addressed each of the scenarios pointed out by the reviewer below:

In our “results section, our opening sentence states “We first tested whether docetaxel is a proconvulsant in C. elegans.” As the reviewer mentioned above, a proconvulsant is a substance that promotes convulsions by lowering seizure threshold in the brain, therefore increasing the risk for seizures. In this study, we did not use docetaxel as a proconvulsant, rather we employed the drug to measure its effects in modulating time-to-recovery from shock-induced seizure-like behaviors. We treated our nematodes in both acute and chronic conditions versus M9 saline to observe for the effects that docetaxel had on the time-to-recovery from the shock-induced seizure-like behaviors. To clarify our methodology and findings, we have reworded the phrasing to state “We first tested whether docetaxel is an agent that can increase time-to-recovery from shock-induced seizure-like behaviors in C. elegans” (Lines 183-184).

Docetaxel is not a proconvulsant but rather a taxane chemotherapeutic agent used to treat advanced, metastatic, or chemotherapy-resistant cancers, however its neurotoxic side effects often lead patients to abandon treatment [5, 6]. We mention this classification for docetaxel in our manuscript (Lines 34, 55, & 58-61). Docetaxel causes docetaxel-induced peripheral neuropathies (DIPNs) which includes motor neuropathy, tingling, muscle weakness, and numbness rather than acute or chronic seizures directly. This re-wording has been made in our manuscript Lines 35-36. Furthermore, in the past research has concluded that seizures following docetaxel use are rare and are not recognized as acute or chronic toxicity of the drug directly. However, a case study published in 1999 did report a patient developing an encephalopathy and subsequent seizures after receiving docetaxel [9]. This leads us to hypothesize that docetaxel may be used as an agent that may sensitize patients to develop seizures. In our study, we used the electroshock assay to model shock-induced DIPN-related muscle weakness C. elegans. We then treated nematodes with acute and chronic conditions of docetaxel to assess the effects of the drug in modulating time to recovery from shock-induced seizure-like behaviors. We have included this clarification in lines 37-39. Results obtained show that acutely or chronically exposing nematodes to docetaxel increases time to recovery from shock-induced seizure-like behaviors (Lines 37-39). Specifically, nematodes treated with docetaxel exposure displayed an increase in time to recovery from shock-induced seizure-like behaviors [Fig1A]. We attribute these drug effects to be concentration dependent, such that after reaching threshold drug toxicity led to the display of increase in behavior duration (Lines 284-289). For chronic docetaxel exposure, we observed increase time to recovery from shcok-induced seizure-like behaviors starting at lower concentrations [Fig2A]. In this case, we attribute drug effects to be time dependent with sensitization, not purely concentration dependent effects (Lines 328-329).

• Line 197 specifically states “docetaxel induces seizure-like behaviors in C. elegans”, which is not supported by the results and needs to be reworded.

We thank the reviewer for pointing this out. Docetaxel does not trigger seizure-like behaviors in our model. Instead it is the shock the one responsible for inducing the seizure-like behavior, while the docetaxel modulates the duration or time to recovery of the seizure-like behavior. We have re-worded this statement throughout the manuscript and we have also re-worded the sentence above (Lines 201-203).

Minor comments:

7. Methods line 94. The day after L4-stage worms would be Day 0 Adulthood (not Day 1).

We revised our used protocol and we found that there was a typo on the days. On day 1, adult worms with eggs were plated on NGM agar plates seeded with OP50 E. coli for 2 days (rather than 3 days) at 20 ℃. On day 3, L4-stage worms were picked and plated on a new NGM agar plate seeded with OP50 E. coli for overnight incubation at 22 ℃. On day 4, 1-day-old adult worms were incubated for 30 minutes in M9 saline (control) or in the respective test solution prior to the electroshock delivery (Lines 99). To stay consistent with our published protocol for the electroshock assay, we stay consistent with the numbering [20].

8. The rationale for some experiments are not well explained. It isn’t clear to this reader why a neuroprotective effect of Viagra was expected. The authors should justify their choice of putative neuro

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PLoS One. doi: 10.1371/journal.pone.0342236.r003

Decision Letter 1

Michael Massiah

16 Jan 2026

Dear Dr. Gonzalez-Lerma,

Thank you for submitting your manuscript to PLOS ONE. As you can see, one of the reviewers still have significant concerns with this manuscript. I hope it is something you can address in a timely manner. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

**********

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The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors have addressed most of my comments; however, I still have a couple of major concerns that should be resolved before publication:

Major Comments

1. That’s fine.

2. Essentially that is fine – and I am certainly not going to require you to redo your controls in light of the provided data. But I would suggest in future always doing your controls in the equivalent volume of solvent. I note in the figure provided that the mean is increasing in 0.1% DMSO (from ~35 to 45 sec) with fairly large error bars. And it may well be that the solvent is a contributing factor to the statistical effect seen in this manuscript.

3-4. I am still unclear on the final concentrations for the chronic exposure experiments and think this needs to be clarified explicitly in the text. From the revised wording, the drugs are dissolved in 1% DMSO and then diluted 1/10 in M9 (equivalent to the acute experiments). For the chronic experiments, this is further dissolved in 10 mls NGM (+ 2 mls solution = 12 mls total). The wording certainly implies that the final DMSO concentrations in the chronic experiments will not be 0.1% - they will be ~0.01%. And it should also be clarified that the final drug concentrations are taking the additional volumes into account as well.

5. This is worded a little better. However, the authors are still implying that “chronic treatment” is different from “acute treatment” by using the phrase “starting at lower concentrations – without overtly saying lower to what? Clearly that is meant to imply lower concentrations to the acute experiments in Figure 1. But there are no statistical comparisons between acute and chronic treatments – indeed the comparisons could not be direct. Your ANOVA in Figure 1 is using more conditions than the ANOVA in Figure 2 and may be affecting the significance. Additionally (see points 3-4 above), I am still unclear that the DMSO concentrations are equivalent for both acute and chronic treatments. I remain suspicious that there is no statistical (let alone biological) effect of chronic treatment in comparison to acute treatment.

6 - 9. That’s fine.

10. The line for acute 0.01 mM docetaxel in Figure 2 should be removed as it is meaningless in the context of the chronic experiment. And indeed the acute 0.01 mM docetaxel from Figure 1A is clearly under 60sec whereas the line in Figure 2A is over 60 sec – so I don’t know where this number is actually generated from?

11. That’s fine.

Reviewer #2: The authors have substantially improved the methods transparency e.g. the number of tubes/condition and the blinding procedures, while also correcting results/legend inconsistency.

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Reviewer #2: No

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PLoS One. 2026 Feb 6;21(2):e0342236. doi: 10.1371/journal.pone.0342236.r004

Author response to Decision Letter 2

19 Jan 2026

Dear Reviewers,

We want to thank the reviewers for their continuous support and constructive feedback. We have thoroughly considered each comment and revised the manuscript accordingly. We believe that the added clarifications and details have continued to strengthen the manuscript. Below, we provide our detailed responses to each point.

Reviewer 1:

Major Comments:

Acute exposure

1. That’s fine.

Thank you for your feedback on this matter and we appreciate that you accept the supporting data we have presented. After revisiting our data and the data previously collected in our lab, we understand the importance of reporting controls that are equivalent to the volume of the solvent used. This methodology indeed clarifies any discrepancies that may arise in future reported data. For future investigations, we will ensure to include controls accordingly.

Chronic exposure

3. & 4. I am still unclear on the final concentrations for the chronic exposure experiments and think this needs to be clarified explicitly in the text. From the revised wording, the drugs are dissolved in 1% DMSO and then diluted 1/10 in M9 (equivalent to the acute experiments). For the chronic experiments, this is further dissolved in 10 mls NGM (+ 2 mls solution = 12 mls total). The wording certainly implies that the final DMSO concentrations in the chronic experiments will not be 0.1% - they will be ~0.01%. And it should also be clarified that the final drug concentrations are taking the additional volumes into account as well.

Thank you for pointing this out. After studying our calculations, we have revised our manuscript to include these specifications in lines 110, and 114-117. We acknowledge that the different DMSO concentrations used for acute versus chronic experiments do not generate a direct comparison between both models.

In our manuscript, we clarify that differences in plate preparation resulted in different DMSO concentrations between chronic and acute exposure experiments (Lines 110, 114-117), making these two methods not directly comparable, as no direct statistical comparison was performed. Additionally, we have changed the description title for Fig 2 to indicate that “Chronic exposure to docetaxel increased time to recovery from shock-induced seizure-like behaviors [Fig 2A] and % NR [Fig 2B], in contrast to nematodes exposed to M9 saline, which did not exhibit such changes” (Lines 205-210 & 332-333). In our Supplemental Figures document titled “S1 Appendix” we have also included this correction (Lines 403-408).

6 - 9. That’s fine.

We have removed the line for acute 0.01 mM docetaxel in Figure 2. This change can be observed in the Figures document submitted with this revision.

11. That’s fine.

Reviewer #2: The authors have substantially improved the methods transparency e.g. the number of tubes/condition and the blinding procedures, while also correcting results/legend inconsistency.

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PLoS One. doi: 10.1371/journal.pone.0342236.r005

Decision Letter 2

Michael Massiah

21 Jan 2026

Anticonvulsant effects of novel and repurposed drugs on docetaxel-induced neuropathy in C. elegans

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Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

**********

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Reviewer #1: Yes

**********

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The PLOS Data policy

Reviewer #1: Yes

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Reviewer #1: Yes

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PLoS One. doi: 10.1371/journal.pone.0342236.r006

Acceptance letter

Michael Massiah

PONE-D-25-51247R2

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. One-way ANOVA: Acute docetaxel treatment increases time to recovery from shock-induced seizure-like behaviors with increasing concentration in C. elegans.

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S2 Fig. Chi-square test: Acute docetaxel treatment increases time to recovery from shock-induced seizure-like behaviors with increasing concentration in C. elegans.

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S5 Fig. One-way ANOVA: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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S6 Fig. Chi-square test: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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pone.0342236.s006.tif^{(267.5KB, tif)}

S7 Fig. One-way ANOVA: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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S8 Fig. Chi-square test: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

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S9 Fig. One-way ANOVA: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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S10 Fig. Chi-square test: Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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S11 Fig. One-way ANOVA: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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pone.0342236.s011.tif^{(226.4KB, tif)}

S12 Fig. Chi-square test: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

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S13 Fig. One-way ANOVA: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors associated with acute and chronic exposure to docetaxel.

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S14 Fig. Chi-square test: Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors associated with acute and chronic exposure to docetaxel.

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S16 Fig. RVM-3 concentration recovery comparisons. Recovery time for N2 worms in M9 saline or in the presence of PTZ.

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Data Availability Statement

All datasets generated and/or analyzed during the current study are available in Open Science Framework DOI: 10.17605/OSF.IO/S56DM.

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PERMALINK

Anticonvulsant effects of novel and repurposed drugs on docetaxel-induced neuropathy in C. elegans

Paola Ximena Gonzalez-Lerma

Crystal Lloyd

Scarlet J Park

Ken Dawson-Scully

Roles

Abstract

Introduction

Materials and methods

C. elegans stocks and cultivation

Acute exposure protocol

Chronic exposure protocol

Drugs and chemicals used

Electroshock assay

Statistical methods

Results

Fig 1. Acute docetaxel treatment increases time to recovery from shock-induced seizure-like behaviors with increasing concentration in C. elegans.

Fig 2. Nematodes treated with chronic docetaxel, display an increase in time to recovery from shock-induced seizure-like behaviors when compared to nematodes exposed to M9 saline alone.

Fig 3. Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

Fig 4. Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors that accompany acute exposure to docetaxel.

Fig 5. Acute treatment with sildenafil citrate decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

Fig 6. Acute treatment with RVM 3 decreases the duration of shock-induced seizure-like behaviors that accompany chronic exposure to docetaxel.

Fig 7. Acute treatment with RVM-3 decreases the duration of shock-induced seizure-like behaviors associated with acute and chronic exposure to docetaxel.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Michael Massiah

Roles

Author response to Decision Letter 1

Decision Letter 1

Michael Massiah

Roles

Author response to Decision Letter 2

Decision Letter 2

Michael Massiah

Roles

Acceptance letter

Michael Massiah

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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