ROCK inhibition with Fasudil induces beta-catenin nuclear translocation and inhibits cell migration of MDA-MB 231 human breast cancer cells

Guerra, Fabiana Sélos; Oliveira, Rogério de; Fraga, Carlos A.M.; Mermelstein, Cláudia; Fernandes, Patrícia Dias

doi:10.1038/s41598-017-14216-z

Cited by 38 publications

(19 citation statements)

References 17 publications

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“…Therefore, as we observed the complete coverage of the scratched areas of the dish in control cells after 24 h of incubation, both compounds, 5b (LASSBio-2020) and 11 (LASSBio-2065), inhibited MDA-MB 231 cell migration. When comparing with data previously obtained by our group showing that fasudil inhibited MDA-MB231 migration, 15 50 µM fasudil reduced cell migration by 50% in the present study. Meanwhile, compounds 5b (LASSBio-2020) and 11 (LASSBio-2065) presented similar effects at lower concentrations (10 µM).…”

Section: Resultssupporting

confidence: 86%

“…Fasudil exerts its effects by upregulating endothelial nitric oxide synthase (eNOS) expression and decreasing smooth muscle spasms and the migration of inflammatory cells 14 . Moreover, as shown in our recent study, the inhibition of ROCK pathway with fasudil induces the nuclear translocation of beta-catenin to reduce MDA MB-231 tumour cell migration 15 .…”

Section: Introductionsupporting

confidence: 64%

“…The compounds were evaluated to determine their potential cytotoxic activities. All compounds were submitted to the MDA-MB-231 cell line viability assay using the MTT quantitative colorimetric method 15 . Relative cell viability was expressed as a percentage relative to control, untreated cells, and none of the evaluated compounds showed toxicity at the tested concentrations at 24 and 48 h. As an example, Figure 8 shows the cell viability at concentrations of compounds 5b (LASSBio-2020) and 11 (LASSBio-2065) ranging from 0.1 to 30 μM.…”

Section: Resultsmentioning

confidence: 99%

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Synthesis and pharmacological evaluation of novel isoquinoline N-sulphonylhydrazones designed as ROCK inhibitors

Oliveira

Guerra

Mermelstein

et al. 2018

Journal of Enzyme Inhibition and Medicinal Chemistry

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In this study, we synthesized a new congener series of N-sulphonylhydrazones designed as candidate ROCK inhibitors using the molecular hybridization of the clinically approved drug fasudil (1) and the IKK-β inhibitor LASSBio-1524 (2). Among the synthesized compounds, the N-methylated derivative 11 (LASSBio-2065) showed the best inhibitory profile for both ROCK isoforms, with IC50 values of 3.1 and 3.8 µM for ROCK1 and ROCK2, respectively. Moreover, these compounds were also active in the scratch assay performed in human breast cancer MDA-MB 231 cells and did not display toxicity in MTT and LDH assays. Molecular modelling studies provided insights into the possible binding modes of these N-sulphonylhydrazones, which present a new molecular architecture capable of being optimized and developed as therapeutically useful ROCK inhibitors.

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Section: Resultssupporting

confidence: 86%

Section: Introductionsupporting

confidence: 64%

Section: Resultsmentioning

confidence: 99%

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Synthesis and pharmacological evaluation of novel isoquinoline N-sulphonylhydrazones designed as ROCK inhibitors

Oliveira

Guerra

Mermelstein

et al. 2018

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…ROCK kinase plays critical roles in tumor metastasis 23 and ROCK inhibitors have shown promising effect on cancer cell migration 24 . ROCK and JAK1 signaling cooperate to control actomyosin contractility in carcinoma-associated fibroblasts to permit the melanoma cell migration 25 .…”

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confidence: 99%

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells

Song

et al. 2020

Sci Rep

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Chemicals and antibody. Celastrol was ordered from MedChemExpress under the item number HY-13067. Antibodies against ezrin, ezrin pT567 and α-tubulin (DM1A) were purchased from Cell Signaling Technology. Antibodies against ROCK2 and ROCK2 (phospho S1366) were purchased from Abcam. Anti-GFP antibodies were purchased from Santa Cruz Biotechnology. Anti-FLAG-tag (M2) antibody was from Sigma. plasmids. The development of bacterial expression vectors containing human ezrin fused to histidine was described previously 61. EGFP-tagged ezrin plasmids was produced as described before 62. PCR amplified ROCK2 cDNA was cloned into the 3× FLAG-Myc-CMV-24 vector (Sigma) by BamHI and RcoRI digestion. The mutanted of ezrin and ROCK2 were constructed by Fast Mutagenesis Kit (Vazyme Biotech). All plasmids were verified by sequencing (Tsingke Biological Tech). cell culture and transfection. MHCC97H was a gift from Professor Yong Chen (Fourth Military Medical University) 20. HepG2 and HEK293T cells were from American Type Culture Collection (ATCC). Huh7 cells were from National Infrastructure of Cell Line Resource. The cells were incubated according to ATCC instruction. Cells were transfected using conventional calcium phosphate method or the Lipofectamine 3,000 (Invitrogen) according to manufacturer's instructions. Determination of cell viability (MTS assay). Celastrol cytotoxicity was assessed with the use of a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega), which is a form of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Cells (5,000 cells/well) were plated in 96-well plates, treated with various concentrations of celastrol for 24 h. After celastrol treatment, cell viability was determined by adding a small amount of the One Solution Reagent directly to culture wells, incubating for 3 h and then recording the absorbance at 490 nm with a 96-well plate reader. Wound healing assay. MHCC97H, HepG2 or Huh7 cells with logarithmic growth phases, was cultivated in 12 orifices with 5 × 10 5 cells density. Each group has three replicates. After a starvation of 6 h in serum-free medium, 10 μl Tip was taken to vertical scratch "#" glyph at the bottom of the culture plate. The floating cells were washed off with PBS and the celastrol was dissolved in medium supplemented with 20% FBS. Samples were photographed at 0 h, 4 h and 8 h. Image J software was used to measure and compare the effects of celastrol on cell migration. Single cell tracking assay. The cell migration by single cell tracking assay was performed as previously described 32. Briefly, MHCC97H cells were laid in a Silian dish. After stabilized, the cells were starved with Opti-MEM for 6 h, and then cultured in DMEM containing 20% FBS. The movement track of 10 single-cells in each field of view were recorded by microscope for 6 h, with 10 min interval at once.

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“…As such, ROCK are generally perceived as key players in cancer development and progression 8 , 19 , which led to consider the use of ROCK inhibitors in the treatment of cancers 7 , 20 , 21 . Indeed, the use of ROCK inhibitors such as Y-27632 or Fasudil (HA-1077) decreased the migration and invasion capacities of several cancer cell types 22 , 23 , among which colon cancer cells 19 , 24 . However, several other studies reported a pro-cancerous effect of ROCK inhibition by promoting the growth and migration of some cancer cells 25 , 26 , such as a gain in colon cancer cell proliferation and invasion 27 – 29 .…”

Section: Introductionmentioning

confidence: 99%

Rock inhibition promotes NaV1.5 sodium channel-dependent SW620 colon cancer cell invasiveness

Poisson

Lopez‐Charcas

Chadet

et al. 2020

Sci Rep

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The acquisition of invasive capacities by carcinoma cells, i.e. their ability to migrate through and to remodel extracellular matrices, is a determinant process leading to their dissemination and to the development of metastases. these cancer cell properties have often been associated with an increased Rho-ROCK signalling, and ROCK inhibitors have been proposed for anticancer therapies. In this study we used the selective ROCK inhibitor, Y-27632, to address the participation of the Rho-ROCK signalling pathway in the invasive properties of SW620 human colon cancer cells. Contrarily to initial assumptions, Y-27632 induced the acquisition of a pro-migratory cell phenotype and increased cancer cell invasiveness in both 3-and 2-dimensions assays. This effect was also obtained using the other ROCK inhibitor Fasudil as well as with knocking down the expression of ROCK-1 or ROCK-2, but was prevented by the inhibition of na V 1.5 voltage-gated sodium channel activity. Indeed, ROCK inhibition enhanced the activity of the pro-invasive na V 1.5 channel through a pathway that was independent of gene expression regulation. In conclusions, our evidence identifies voltage-gated sodium channels as new targets of the ROCK signalling pathway, as well as responsible for possible deleterious effects of the use of ROCK inhibitors in the treatment of cancers.

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ROCK inhibition with Fasudil induces beta-catenin nuclear translocation and inhibits cell migration of MDA-MB 231 human breast cancer cells

Cited by 38 publications

References 17 publications

Synthesis and pharmacological evaluation of novel isoquinoline N-sulphonylhydrazones designed as ROCK inhibitors

Synthesis and pharmacological evaluation of novel isoquinoline N-sulphonylhydrazones designed as ROCK inhibitors

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells

Rock inhibition promotes NaV1.5 sodium channel-dependent SW620 colon cancer cell invasiveness

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