Effect of Cichoric Acid on Colorectal Cancer: Impact on Migration, Epithelial-Mesenchymal Transition, and Proliferation via RhoA/ROCK Signaling Pathway Modulation.

BACKGROUND: Colorectal cancer (CRC) is a common malignancy with high morbidity and mortality. To improve CMC prognosis, research must identify safe and effective natural drugs that improve the proliferation, migration, and epithelial mesenchymal transition (EMT) processes of CRC. The purpose of this paper is to understand how cichoric acid (CA) impacts CRC proliferation, metastasis, and EMT of CRC by adjusting the Ras homolog family member A (RhoA)/RHO-associated coiled coil protein kinase (ROCK) pathway. METHODS: Human Colon Cancer Cells (HCT116) cells were randomly divided into Control (blank medium treatment), low concentration CA (CA-L), medium concentration CA (CA-M), high concentration CA (CA-H), and high-concentration CA+RhoA activator U46619 (CA-H+U46619) groups. Cell proliferation, migration and invasion, and apoptosis were evaluated with cell counting kit-8 (CCK-8) assay, transwell assay, and flow cytometry, respectively. The expression of RhoA, ROCK, and EMT-associated proteins were detected by Western Blot. The CRC transplanted tumor model of nude mice was constructed, and the mice were grouped into low-dose CA (CA-Low, 15 mg/kg CA), high-dose CA (CA-High, 30 mg/kg CA), high-dose CA+RhoA activator U46619 (CA-High+U46619, 30 mg/kg CA+10 mM U46619), and Model groups at random, with 12 mice in each group. Tumor volume, mass, and inhibition rate were measured and calculated, and the pathological changes of tumor in nude mice were detected by hematoxylin-eosin (HE) staining. RESULTS: Compared with Control, the optical density of cells at 450 nm (OD450) value (48 h, 72 h), cell migration number, cell invasion number, RhoA, ROCK1, N-cadherin, vimentin protein expression levels of HCT116 cells were reduced in CA-M and CA-H groups; however, E-cadherin level and apoptosis rate were increased (p < 0.05). In the CA-High group, we observed a significant decrease (p < 0.05) in both tumor volume and mass in nude mice. Additionally, the tumor tissue cells exhibited better organization, reduced size, reduced tumor and vascular tissue hyperplasia, and decreased infiltration of inflammatory cells. U46619 decreased the retardation of CA on the proliferation, EMT, and migration of CRC tumor cells as well as the growth of transplanted CRC tumors in nude mice. CONCLUSIONS: CA may reduce CRC migration, proliferation, and EMT by inhibiting the activation of the RhoA/ROCK signaling pathway.

Preclinical Evidence that Arctigenin Effectively and Selectively Targets Clear Cell Renal Cell Carcinoma Via Suppressing EGFR and RhoA.

Clear cell renal cell carcinoma (ccRCC) has poor clinical outcomes and necessitates new treatment options. Epidermal growth factor receptor (EGFR) is a potential therapeutic target, due to the associations with various carcinomas' progression. Arctigenin, a natural compound of Arctium lappa, has been shown to display anticancer abilities in various carcinomas. Cellular assays and combination studies were conducted using arctigenin and anti-ccRCC drugs. In vivo efficacy of arctigenin was determined using ccRCC xenograft mouse model. Immunoblotting and biochemistry analysis were applied to investigate the signaling affected by arctigenin. Arctigenin inhibits growth, migration, and survival of ccRCC cells while sparing normal kidney cells. Arctigenin acts synergistically with 5-FU and sorafenib but not temsirolimus in inhibiting ccRCC cells. Synergism of arctigenin with 5-FU and sorafenib was further shown in ccRCC xenograft mouse model. The combination of arctigenin with clinical anti-RCC drugs completely inhibits tumor growth without tumor progression even for an extended time period. Mechanistically, arctigenin inhibits migration in a RhoA-dependent manner while inhibits growth via suppressing EGFR-mediated signaling pathways. Our findings suggest that arctigenin performs well to add to current treatment in ccRCC and confirm the value to target EGFR to improve therapy in RCC.

Ulinastatin ameliorates the malignant progression of prostate cancer cells by blocking the RhoA/ROCK/NLRP3 pathway.

Prostate cancer is a male malignant tumor disease with high incidence and mortality. This study was designed to explore the effects of ulinastatin (UTI) on the malignant progression of prostate cancer and its relevant mechanism of action. Human prostate cancer cell line PC-3 was applied to investigate the anticancer activity of UTI. PC-3 cells were treated with increasing concentrations (400, 800, and 1600 U/ml) of UTI. Cell proliferation, migration, invasion, and apoptosis were determined by cell counting kit-8 (CCK-8), colony formation, wound-healing, Transwell assay, and flow cytometry analysis, respectively. The expression level of corresponding proteins was detected by western blot. In addition, PC-3 cells were pretreated with RhoA agonist CN03 (1 µg/ml) or NLRP3 agonist nigericin (10 µM) before UTI treatment, and the cellular behaviors above were detected again. It was demonstrated that UTI significantly suppressed cell proliferation, migration, and invasion but promoted apoptosis in PC-3 cells in a concentration-dependent manner. Meanwhile, UTI could block RhoA/ROCK/NLRP3 inflammasome pathway in PC-3 cells, and the activation of RhoA or NLRP3 inflammasome partly weakened the impacts of UTI on cell proliferation, migration, and apoptosis in PC-3 cells, respectively. In summary, our study demonstrated the antitumor activity of UTI against prostate cancer by regulating RhoA/NLRP3 inflammasome pathway, providing a promising candidate drug for the therapeutic treatment of prostate cancer.

Monensin synergizes with chemotherapy in uveal melanoma through suppressing RhoA.

OBJECTIVE: Uveal melanoma (UM) is the common primary cancer of the eye and new treatments are needed. Substantial evidence has shown that an antibiotic monensin is an attractive candidate for the development of anti-cancer drug. In this study, we investigated the potential of repositioning monensin for the treatment of UM in the pre-clinical setting. MATERIALS AND METHODS: Cellular activity assays were performed using multiple cell lines representing UM models with different cellular origins and genetic profiling and normal cells as control. Combination studies were performed using Chou-Talalay method. Mechanism studies were performed using immunoblotting and ELISA. RESULTS: Monensin was effective against all tested UM cell lines and less effective against normal fibroblast cells. Monensin induced G0/G1 arrest and thus decreased S phase, leading to UM cell growth inhibition. It also inhibited migration and induced apoptosis in UM cells. In addition, the combination of monensin and dacarbazine was synergistic in targeting UM cells. Our mechanistic studies showed that monensin specifically decreased activity of RhoA without affecting other small GTPases, such as Ras and Rac1. Consistently, monensin decreased phosphorylation of downstream effectors of RhoA signaling, including ROCK, MYPT1 and MLC. Rescue studies using RhoA activator calpeptin showed that calpeptin significantly abolished the inhibitory effects of monensin on RhoA activity, proliferation, migration and survival, confirming that RhoA is the target of monensin in UM cells. CONCLUSIONS: Our study demonstrates that monensin is a potent inhibitor of UM and synergizes with chemotherapy, via suppressing RhoA activity and RhoA-mediated signaling. Our findings suggest that monensin may be a potential lead compound for further development into a drug for UM treatment.

RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells: implications for Duchenne muscular dystrophy.

BACKGROUND: The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF-AA have not been described so far. Our aim was to study the molecular role of PDGF-AA in the fibrotic process of DMD. METHODS: Skeletal muscle fibro-adipogenic progenitor cells (FAPs) from three DMD treated with PDGF-AA at 50 ng/mL were analysed by quantitative mass spectrometry-based proteomics. Western-blot, immunofluorescence, and G-LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF-AA on the activation of RhoA pathway using two inhibitors, C3-exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J-mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fibrotic and inflammatory markers in muscle tissue. RESULTS: Mass spectrometry revealed that RhoA pathway proteins were up-regulated in treated compared with non-treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7-fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF-AA (n = 3, 1.88-fold increase, P < 0.01) and both C3-exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF-AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3-exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, 1.76-fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen-I expression area (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, P < 0.01). CONCLUSIONS: Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies.

Rho kinase inhibition ameliorates vascular remodeling and blood pressure elevations in a rat model of apatinib-induced hypertension.

OBJECTIVES: Hypertension is one of the major adverse effects of tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factors. However, the mechanism underlying TKIs-induced hypertension remains unclear. Here, we explored the role of the RhoA/Rho kinase (ROCK) signaling pathway in elevation of blood pressure (BP) induced by apatinib, a selective TKI approved in China for treatment of advanced or metastatic gastric cancer. A nonspecific ROCK inhibitor, Y27632, was then combined with apatinib and its efficacy in alleviating apatinib-induced hypertension was evaluated. METHODS: Normotensive female Wistar-Kyoto rats were exposed to two different doses of apatinib, or apatinib combined with Y27632, or vehicle for 2 weeks. BP was monitored by a tail-cuff plethysmography system. The mRNA levels and protein expression in the RhoA/ROCK pathway were determined, and vascular remodeling assessed. RESULTS: Administration of either a high or low dose of apatinib was associated with a rapid rise in BP, reaching a plateau after 12 days. Apatinib treatment mediated upregulation of RhoA and ROCK II in the mid-aorta, more significant in the high-dose group. However, ROCK I expression showed no statistically significant differences. Furthermore, the mRNA level of GRAF3 decreased dose-dependently. Apatinib administration was also associated with decreased levels of MLCP, and elevated endothelin-1 (ET-1) and collagen I, which were accompanied with increased mid-aortic media. However, treatment with Y27632 attenuated the above changes. CONCLUSION: These findings suggest that activation of the RhoA/ROCK signaling pathway could be the underlying mechanism of apatinib-induced hypertension, while ROCK inhibitor have potential therapeutic value.

Inhibition of respiratory syncytial virus by RhoA-derived peptides: implications for the development of improved antiviral agents targeting heparin-binding viruses.

The respiratory syncytial virus (RSV) fusion glycoprotein (F) can interact with the small intracellular GTPase RhoA, and peptides derived from RhoA inhibit RSV replication. These observations initially suggested that RhoA-derived peptides might inhibit RSV replication by disrupting an in vivo interaction between RSV F and RhoA. However, recent data indicate that the antiviral activity of RhoA-derived peptides is not due to competitive inhibition of an hypothesized F-RhoA interaction, but is rather a function of the peptides' intrinsic biophysical properties. We summarize here what is known about the mechanism of RSV inhibition by these peptides and give our opinion regarding the potential implications of this work with regards to RSV biology, and to the development of antiviral agents targeting RSV and other enveloped viruses.