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.
Cancer-type Organic anion transporting polypeptide 1B3, ct-OATP1B3-V1 is a tumor-specific isoform of liver-type OATP1B3 (Lt-OATP1B3). Ct-OATP1B3-V1 is identical with liver-specific Lt-OATP1B3 except it lacks the first 28 amino acids. Although there is a growing interest in using this isoform as a biomarker for colorectal cancer, available data regarding cellular localization and function of ct-OATP1B3-V1 remains controversial. The main objective of our study was to clarify the localization and function of ct-OATP1B3-V1 in vitro and in vivo, and to investigate its role in chemotherapy sensitivity. For this aim, A431 and HCT-8 carcinoma cell lines overexpressing ct-OATP1B3-V1 were generated. With the help of these cell lines, localization and activity of ct-OATP1B3-V1 as well as its effect on chemotherapy sensitivity was examined both in vitro and in vivo. We found that ct-OATP1B3-V1 is a functional plasma membrane transporter that sensitizes the cells toward various chemotherapeutics, including docetaxel, oxaliplatin and capecitabine metabolites in vitro. Increased sensitivity to docetaxel and capecitabine of ct-OATP1B3-V1 expressing cells was also confirmed in in vivo experiments performed on A431-V1 derived xenografts. However, due to the apparent proliferative advantage of V1-expressing xenografts over the mock-transfected control, they could not be completely eradicated by either docetaxel or capecitabine treatment. Our results demonstrate that while ct-OATP1B3-V1 can be exploited to inhibit tumor growth, this strategy alone is likely insufficient for complete tumor elimination, possibly due to the more complex in vivo functions of ct-OATP1B3-V1.
This study leverages physiologically based biopharmaceutics modeling (PBBM) to predict the clinical performance of two itraconazole (ITRA) amorphous solid dispersions (ASDs), Sempera® and Tolsura®, under fasted and fed state conditions, exploring the potential of PBBM in predicting formulation-specific food interactions. The ITRA formulations were subjected to extensive in vitro biopharmaceutical testing, including solubility studies and dissolution tests under fasted and fed state conditions, revealing significant differences in dissolution behaviors between Sempera® and Tolsura®. The impact of food and hypochlorhydria on drug absorption was evaluated using a stepwise mechanistic deconvolution-reconvolution PBBM approach, integrating fundamental parameters based on the in vitro data into the final model. Our model not only successfully predicted the effects of acid reducing agents (ARA) and food on the oral absorption of ITRA, but also captured the between-subject variability, demonstrating the utility of this approach in understanding the complex interplay between drug, formulation, and gastrointestinal environment. Most importantly, the PBBM was able to accurately predict the positive impact of food on the absorption of Sempera® and the negative food effect of Tolsura®. The findings highlight the importance of considering formulation characteristics and gastrointestinal physiology, underscoring the potential of PBBM in bioequivalence (BE) assessment of generic formulations under varying physiological conditions, including in the fed state and in hypochlorhydric patients. The successful application of this stepwise and mechanistic PBBM approach suggests a potential pathway for streamlining drug development and may contribute to more informed decision-making for BE assessment.