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. 2024 Jan 1;165(1):54-74.
doi: 10.1097/j.pain.0000000000002972. Epub 2023 Jun 27.

Chemotherapy for pain: reversing inflammatory and neuropathic pain with the anticancer agent mithramycin A

Zheyun Xu  1 , Man-Cheung Lee  1 , Kayla Sheehan  1 , Keisuke Fujii  1   2 , Katalin Rabl  1 , Gabriella Rader  1 , Scarlett Varney  1 , Manohar Sharma  1 , Helge Eilers  1 , Kord Kober  3 , Christine Miaskowski  3 , Jon D Levine  4 , Mark A Schumacher  1
Affiliations

Chemotherapy for pain: reversing inflammatory and neuropathic pain with the anticancer agent mithramycin A

Zheyun Xu et al. Pain. .

Abstract

The persistence of inflammatory and neuropathic pain is poorly understood. We investigated a novel therapeutic paradigm by targeting gene networks that sustain or reverse persistent pain states. Our prior observations found that Sp1-like transcription factors drive the expression of TRPV1, a pain receptor, that is blocked in vitro by mithramycin A (MTM), an inhibitor of Sp1-like factors. Here, we investigate the ability of MTM to reverse in vivo models of inflammatory and chemotherapy-induced peripheral neuropathy (CIPN) pain and explore MTM's underlying mechanisms. Mithramycin reversed inflammatory heat hyperalgesia induced by complete Freund adjuvant and cisplatin-induced heat and mechanical hypersensitivity. In addition, MTM reversed both short-term and long-term (1 month) oxaliplatin-induced mechanical and cold hypersensitivity, without the rescue of intraepidermal nerve fiber loss. Mithramycin reversed oxaliplatin-induced cold hypersensitivity and oxaliplatin-induced TRPM8 overexpression in dorsal root ganglion (DRG). Evidence across multiple transcriptomic profiling approaches suggest that MTM reverses inflammatory and neuropathic pain through broad transcriptional and alternative splicing regulatory actions. Mithramycin-dependent changes in gene expression following oxaliplatin treatment were largely opposite to and rarely overlapped with changes in gene expression induced by oxaliplatin alone. Notably, RNAseq analysis revealed MTM rescue of oxaliplatin-induced dysregulation of mitochondrial electron transport chain genes that correlated with in vivo reversal of excess reactive oxygen species in DRG neurons. This finding suggests that the mechanism(s) driving persistent pain states such as CIPN are not fixed but are sustained by ongoing modifiable transcription-dependent processes.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Figure 1.
Figure 1.
MTM reverses CFA- and cisplatin-induced thermal (heat) hyperalgesia. (A) Hindpaw injection of CFA induced inflammatory heat hyperalgesia (decrease in thermal paw withdrawal latency) at 24 and 48 hours and was reversed to baseline control values in male mice after MTM treatment (100 µg/kg ip,—bars) on day 6 (*P = 0.028) and day 10 (**P = 0.0025); n = 17. MTM did not change heat thresholds when given alone, n = 10. (B) An increase in CFA-induced hindpaw thickness (inflammation) was unchanged by MTM treatment (ns = not significant). (C) Comparison of CFA-induced inflammatory heat hyperalgesia between female and male mice. Despite equivalent baseline latencies, female mice developed a greater CFA-induced decrease in latency (heat hyperalgesia) compared with male mice on days 1 (**P = 0.001), 6 (****P < 0.0001), and 10 (**P = 0.001); multiple t tests; n = 11. (D) MTM directed reversal of heat hyperalgesia in CFA-treated female mice on days 6 (**P = 0.007) and 10 (***P = 0.0001); multiple t tests; n = 14 but was less than that found in MTM-treated male mice on days 6 (**P = 0.007) and 10 (***P = 0.0004); multiple t tests; n = 11. MTM-treated female mice attained latency values observed for untreated CFA-male mice on days 6 and 10, whereas MTM-treated male mice recovered to baseline values on the same time points. (E) Cisplatin (Cis) (3 mg/kg ip) administered daily for 5 days decreased heat paw withdrawal latency at 24 hours. Paw withdrawal latency was reversed to baseline values following MTM treatment starting on day 3 through day 14 (****P < 0.0001; ***P = 0.0009); n = 6. (F) Cisplatin treatment (5 days)-induced mechanical hypersensitivity (decrease in mechanical threshold) beginning at 24 hours was reversed following MTM treatment to baseline values by day 7 through day 21 (****P < 0.0001); n = 6. Statistical comparison: 2-way RM ANOVA with Tukey post hoc test. Mean values ± SEM. CFA, complete Freund adjuvant; MTM, mithramycin; RM ANOVA, repeated-measures analysis of variance; Veh, vehicle.
Figure 2.
Figure 2.
MTM reverses oxaliplatin-induced cold and mechanical hypersensitivity. (A) Oxaliplatin (OX) (3 mg/kg ip) induced a decrease in paw withdrawal latency (cold hypersensitivity) at 48 hours (***P = 0.0005) and 72 hours (****P < 0.0001). Reversal of cold hypersensitivity was achieved at 72 hours following MTM treatment 10 µg/kg, 50 µg/kg, and more so at 100 µg/kg (*P < 0.05, ns = not significant); n = 4. (B) Longitudinal study of MTM (100 µg/kg ip,—bars) reversed the oxaliplatin-induced cold hypersensitivity on days 3, 6, 10, 14, and 21 (*P = 0.022, ****P < 0.0001); n = 6. Control oxaliplatin-treated mice maintained cold hypersensitivity through day 21. MTM alone did not change paw withdrawal latency to 10°C. (C) Testing was repeated at 4°C demonstrating reversal on days 3 (**P = 0.0083), 6 (****P < 0.0001), 10 (**P = 0.0016), 14 (*P = 0.016), and 21 (****P < 0.0001); n = 6. (D) Oxaliplatin treatment induced a decreased mechanical threshold (mechanical hypersensitivity) at 24 and 48 hours that was reversed on days 3, 6, 10, and 14 following MTM treatment (bar) (*P = 0.044, ****P < 0.0001); n = 6. MTM did not change mechanical thresholds when given alone. (E to G) Oxaliplatin-treated female mice following MTM-rescue showed limited ability to reverse cold (4°C) hypersensitivity on days 3 (**P = 0.0052) or mechanical hypersensitivity on day 10 (P = 0.051) and day 21 (P = 0.053); n = 6. Statistical comparison: 2-way RM ANOVA with Tukey post hoc test. Mean values ± SEM. MTM, mithramycin; RM ANOVA, repeated-measures analysis of variance; Veh, vehicle.
Figure 3.
Figure 3.
MTM reversal of oxaliplatin-induced cold hypersensitivity is linked to TRPM8 expression in DRG neurons. (A) Percentage of DRG neurons responding to cold buffer (10.5-12.1°C; mean 11.3°C) derived from mice treated in vivo with vehicle (Veh), oxaliplatin (OX) (3 mg/kg), or OX + MTM (100 µg/kg) as conducted in Figure 2B and harvested on day 4. MTM reversed the increased number of cold-responding DRG neurons ex vivo. n = 3 independent trials, total neurons = 257 (Fisher exact test *P = 0.019, ***P = 0.0008, ns = not significant). (B) Size distribution histogram of cold responding DRG neurons (black, n = 47) were of smaller size than nonresponders (grey, n = 210) (P < 0.0001 unpaired t test, Welch correction). (C) DRG TRPM8 mRNA expression (RQ) was increased approximately 4 fold following oxaliplatin treatment in vivo. MTM (100 µg/kg) reversed the OX-induced increase in DRG TRPM8 mRNA expression to baseline levels (1-way ANOVA with Tukey post hoc test (**P = 0.001; ***P = 0.0006); n = 5, triplicate measures. mean ± SEM. (D) Validation of TRPA1 antagonist HC-030031 to block AITC-induced paw licking. Mice received HC-030031 (100 mg/kg i.p.) at 2 hours. Postantagonist, 0.1% AITC (20 µL, ipl, in saline), was administered. AITC-elicited licking behaviors(s) were completely blocked by HC-030031 (*P = 0.015); n = 6. Unpaired t test. (E) Validation of TRPM8 antagonist AMG333 to block icilin-induced wet dog shakes (WDS). Mice administered AMG333 (3 mg/kg, p.o), and 2 hours later, icilin (20 mg/kg ip) completely blocked icilin-induced WDS (**P = 0.0085); n = 8. Unpaired t test. (F) Mice were treated with OX on day 0 and developed cold hypersensitivity (decreased withdrawal latency to 10°C) on day 6, T0, TRPM8 antagonist AMG333 reversed the cold hypersensitivity 2 hours post the administration on day 6, T2 (**P = 0.002) n = 7 to 9. Statistical comparison: 2-way ANOVA. Mean ± SEM. Administration of TRPA1 antagonist HC-030031 failed to reverse OX-induced cold hypersensitivity. AITC, allyl isothiocyanate; ANOVA, analysis of variance; DRG, dorsal root ganglion; MTM, mithramycin.
Figure 4.
Figure 4.
MTM reverses persistent oxaliplatin-induced cold and mechanical hypersensitivity. (A) Oxaliplatin (3 mg/kg ip) twice weekly for 4 weeks induced a persistent reduction in paw withdrawal latency to 10°C (cold hypersensitivity). MTM (100 µg/kg ip—bar) given on days 28 and 29 reversed cold hypersensitivity to near baseline values on days 29 to 35 (***P < 0.0001); n = 6. (B) MTM reversed oxaliplatin-induced hypersensitivity to 4°C on days 29 (***P = 0.0004), 30 (**P = 0.0011), 31 (*P = 0.049), and 35 (*P = 0.025); n = 6. (C) MTM produced a reversal of oxaliplatin-induced mechanical hypersensitivity on days 29 to 35 (***P < 0.0001); n = 6. There was no change in cold withdrawal latency or mechanical thresholds following MTM treatment alone. Statistical comparison: 2-way RM ANOVA with Tukey post hoc test. MTM, mithramycin; OX, oxaliplatin; RM, repeated-measures analysis of variance; Veh, vehicle.
Figure 5.
Figure 5.
Intraepidermal nerve fiber (IENF) density of mouse skin following longer-term oxaliplatin administration with or without MTM rescue. Representative fluorescent images (arrows) of IENF visualized in mouse skin with anti-PGP9.5 targeted immunofluorescence (red) overlayed with corresponding bright field images (40X). (A) Vehicle-injected mice. (B) Mice treated twice weekly × 4 weeks with oxaliplatin. (C) Mice treated twice weekly with oxaliplatin × 4 weeks followed by MTM (100 µg/kg ip) on days 28 and 29. (D) Mice treated twice weekly with vehicle only × 4 weeks followed by MTM on days 28 and 29. Administration of oxaliplatin, MTM, and vehicle were exactly as described in Figure 4. Scale bars are 100 µm. (E) Quantitation of IENF density shows a decrease in IENF between OX vs Veh (*P < 0.05), OX vs MTM (**P = 0.003), OX + MTM vs MTM alone (****P < 0.0001), and OX + MTM vs Veh (**P = 0.002). Oxaliplatin (OX) = 3 mg/kg ip. MTM = 100 µg/kg ip. Image analysis is derived from 5 random sections from each mouse (n = 3) using 3 fields of view per sectionP. Statistical comparison: ANOVA with Tukey post hoc test. Mean ± SEM. ANOVA, analysis of variance; MTM, mithramycin; OX, oxaliplatin; Veh, vehicle.
Figure 6.
Figure 6.
Oxaliplatin-induced differentially expressed genes and enriched pathways in DRG. (A) Top differentially expressed genes (DEGs) with vehicle only (vehicle + vehicle = VV) as base, compared with oxaliplatin-only (oxaliplatin + vehicle) treatment. Downregulated (blue) and upregulated (red) DEGs are shown. FDR 0.38 (fold change greater than 1.5). (B) Enrichment analysis of OX-treated DRG (Metascape) using ontology sources: GO Biological Processes, KEGG Pathway, Reactome Gene Sets, CORUM, PaGenBase, WikiPathways, and PANTHER Pathway. Log10(P) is the P value in log base 10. (C) Enrichment analysis of OX-treated DRG by TRRUST (Transcriptional Regulatory Relationships Unraveled by Sentence – based Text mining (https://www.grnpedia.org/trrust/). DRG, dorsal root ganglion.
Figure 7.
Figure 7.
Transcriptional changes in DRG because of oxaliplatin and MTM treatment. (A) Venn diagram showing differentially expressed genes (DEGs) (FDR
Figure 8.
Figure 8.
Mithramycin-rescue of differentially expressed genes and enriched pathways in DRG. (A) Top differentially expressed genes (DEGs) with oxaliplatin-only (oxaliplatin + vehicle = OV) as base, compared with oxaliplatin + mithramycin (OM) treatment. Downregulated (blue) and upregulated (red) DEGs are shown. FDR 0.38 (fold change greater than 1.5). (B) Enrichment analysis of MTM-rescue DRG (Metascape) using ontology sources: GO Biological Processes, KEGG Pathway, Reactome Gene Sets, CORUM, PaGenBase, WikiPathways and PANTHER Pathway. Log10(P) is the P value in log base 10. (C) Enrichment analysis of MTM-rescue DRG by TRRUST (Transcriptional Regulatory Relationships Unraveled by Sentence–based Text mining (https://www.grnpedia.org/trrust/). DRG, dorsal root ganglion; MTM, mithramycin.
Figure 9.
Figure 9.
Model of primary afferent nociceptor expressing differentially expressed genes (DEGs) identified from MTM rescue of oxaliplatin-induced hypersensitive state. The DRG MTM treatment group (OM) was used as input for database mining to ascribe a putative categorization from publicly available pain gene databases including previous studies,, and the Pain Research Forum - Pain Gene Resource and automated text-based Medline searches (easyPubmed package in R). Downregulated (blue) and upregulated (red) genes are shown. DRG, dorsal root ganglion; MTM, mithramycin.
Figure 10.
Figure 10.
Components of DRG mitochondrial electron transport chain (ETC) are negatively regulated by MTM rescue of oxaliplatin-induced hypersensitivity. Protein–protein interaction enrichment analysis (Metascape) of oxaliplatin (OX) + MTM using databases: STRING, BioGrid, OmniPath, InWeb IM, and the Molecular Complex Detection (MCODE) algorithm, identified MCODE module 5 (ETC) as the best scoring terms by P value (see boxed values for ETC module Log10P). DRG, dorsal root ganglion; MTM, mithramycin.
Figure 11.
Figure 11.
Mithramycin (MTM) reverses in vivo oxaliplatin-induced DRG neuronal production of reactive oxygen species (ROS). Normalized ROS fluorescence (ROS cell mean GFP * ROS cell #)/(Cell mean GFP * Cell #) ex vivo measured from DRG neurons cultured on day 10 following in vivo treatment of mice with Ox + Veh (3 mg/kg, i.p.) on day 0, MTM + Veh (100 µg/kg, i.p.) on day 2 and day 3 and harvested on day 10. MTM reversed the OX-induced 2.2-fold increase in ROS in DRG neurons (*** P = 0.0004; ns = not significant); n = 3 independent experiments, each with 3 replicate culture. Statistical comparison: ordinary one-way ANOVA, Tukey post hoc test. Mean values ± SEM. ANOVA, analysis of variance; DRG, dorsal root ganglion.
Figure 12.
Figure 12.
Mithramycin (MTM) rescue of oxaliplatin-induced hypersensitivity reveals a unique cohort of DRG spliced genes. (A) Principal component analysis (PCA) plot of splice events across 1533 OM and OV genes is shown. Red indicates MTM rescue group, and blue indicates OX treatment only. Differential alternative splicing analysis performed by RMAT. (B) Enrichment analysis identified differentially spliced genes dedicated to cytoskeleton intracellular transport and synaptic regulation as top pathways associated with MTM rescue in DRG. Databases (Metascape): GO Biological Processes, KEGG Pathway, Reactome Gene Sets, CORUM, PaGenBase, WikiPathways, and PANTHER Pathway. Log10(P) is the P-value in log base 10. DRG, dorsal root ganglion.
Figure 13.
Figure 13.
Hypothetical mechanism of mithramycin's (MTM’s) reversal of oxaliplatin-induced painful hypersensitivity. (A) Oxaliplatin (platinum - Pt) binds to dorsal root ganglion (DRG) nuclear and mitochondrial DNA forming intrastrand adducts. Oxaliplatin-induced changes in DNA structure allow binding of repressor proteins (left) to genes contributing to antinociception and activation of pronociceptive genes driven by GC box–binding transcription factors Spx such as Sp1 and Sp4. Oxaliplatin-induced transcriptional changes in sensory neurons result in 930 differentially expressed genes (DEGs), a subset of which drive painful hypersensitivity to heat, mechanical and cold stimuli. Oxaliplatin induces an increase in DRG intracellular reactive oxygen species (ROS) known to elicit pain, in part, by activating transient potential (TRP) channels, such as TRPM8, shown to mediate cold hypersensitivity. (B) Mithramycin (MTM) binding to GC-rich DNA regulatory domains under conditions of oxaliplatin-induced DNA modification is proposed to reverse oxaliplatin-induced painful hypersensitivity through the inhibition of Spx transcription factor binding and by a combination of derepression of antinociceptive genes such as Mt2 metallothionein and Nts neurotensin (left) and repression of pronociceptive genes such as TRPM8 (right). Changes in 496 DEGs include modulation of genes encoding Complex 1 of the mitochondrial electron transport chain are proposed to contribute to the normalization of ROS levels in oxaliplatin-treated DRG. (C) MTM treatment alone does not significantly bind or alter DRG DNA structure and thus does not change sensory threshold withdrawal values to noxious heat, mechanical or cold stimuli, nor evoke a change in DRG ROS levels or TRPM8 expression. The small number of MTM-only DEGs (22) likely reflect the limited ability of MTM to access GC-binding domains under conditions without oxaliplatin-induced structural changes.
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