Exploring the Novel Role and Mechanistic Insights of Skeletal Muscle Relaxant Cyclobenzaprine Hydrochloride in Esophageal Squamous Cell Carcinoma Treatment.

Abstract

Objective: Cyclobenzaprine hydrochloride (Flexeril) is a muscle relaxant primarily used to relieve muscle pain and spasms. However, its potential anti-cancer role remains largely unexplored. This study aims to investigate the inhibitory effect of Flexeril on esophageal squamous cell carcinoma (ESCC) and to uncover the molecular mechanisms through which it affects the proliferation and metastasis of ESCC.

Methods: A compound library approved by the FDA was employed to screen drugs with inhibitory effects on ESCC. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay, and Plate colony formation was analyzed to evaluate the proliferative ability of ESCC cell lines (KYSE150 and Eca109) after treatment with Flexeril. Migratory ability was examined through Transwell and Scratch assays. Proteomics was performed to identify proteins regulated by Flexeril in KYSE150 and Eca109 cells. RT-PCR and Western blot were used to detect changes in related genes at the mRNA and protein levels after treatment with Flexeril. Drug affinity responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA) were employed to identify the binding of Flexeril and JAK1 protein. Additionally, the comet assay was conducted to assess the DNA damage response in ESCC cells following WDHD1 knockdown or Flexeril exposure. Finally, tumor‑bearing nude mice model were constructed to evaluate the in vivo anticancer effects of Flexeril on ESCC.

Results: Flexeril significantly inhibited the proliferation and migration of ESCC cells in a time- and dose-dependent manner. Proteomics analysis identified WDHD1 as a downstream target of Flexeril exposure, and knockdown of WDHD1 mimicked the effects of Flexeril on proliferation and migration of ESCC. Conversely, overexpression of WDHD1 attenuated the inhibitory effects of Flexeril on ESCC. Mechanistically, the JAK1-STAT3 signaling pathway, but not the JAK2-STAT3 or PI3K-Akt-mTOR pathways, was involved in regulating WDHD1 expression in ESCC cells following Flexeril treatment. Overexpression of STAT3 or WDHD1 mitigated the inhibitory effects of Flexeril on ESCC proliferation and migration. Moreover, both Flexeril exposure and WDHD1 knockdown induced a DNA damage response (DDR) in ESCC cells. In addition, Flexeril significantly inhibited the growth of ESCC tumors in nude mice, downregulating the JAK1-STAT3-WDHD1 signaling pathway, with no significant damage observed in vital organs such as the heart, liver, spleen, lungs, or kidneys, as shown by histological examination.

Conclusion: Flexeril exhibits anti-cancer effects in ESCC by inhibiting the JAK1-STAT3-WDHD1 axis and inducing DDR. These findings suggest that Flexeril may serve as a potential novel therapeutic agent for the treatment of ESCC.