Epithelial-mesenchymal transition (EMT) in primary tumor cells is a key prerequisite for metastasis initiation. Statins, cholesterol-lowering drugs, can delay metastasis formation in vivo and attenuate the growth and proliferation of tumor cells in vitro. The latter effect is stronger in tumor cells with a mesenchymal-like phenotype than in those with an epithelial one. However, the effect of statins on epithelial cancer cells treated with EMT-inducing growth factors such as transforming growth factor-β (TGF-β) remains unclear. Here, we examined the effect of atorvastatin on two epithelial cancer cell lines following TGF-β treatment. Atorvastatin-induced growth inhibition was stronger in TGF-β-treated cells than in cells not thusly treated. Moreover, treatment of cells with atorvastatin prior to TGF-β treatment enhanced this effect, which was further potentiated by the simultaneous reduction in the expression of the statin target enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Dual pharmacological targeting of HMGCR can thus strongly inhibit the growth and proliferation of epithelial cancer cells treated with TGF-β and may also improve statin therapy-mediated attenuation of metastasis formation in vivo.

The epithelial‐to‐mesenchymal transition (EMT) is fundamental in cancer progression and contributes to the acquisition of malignant properties. The statin class of cholesterol‐lowering drugs exhibits pleiotropic anticancer effects in vitro and in vivo, and many epidemiologic studies have reported a correlation between statin use and reduced cancer mortality. We have shown previously that sensitivity to the anti‐proliferative effect of statins varies among human cancer cells and statins are more effective against mesenchymal‐like cells than epithelial‐like ones in human cancers. There have only been few reports on the application of statins to cancer therapy in veterinary medicine, and differences in statin sensitivity among canine cancer cells have not been examined. In this study, we aimed to clarify the correlation between sensitivity to atorvastatin and epithelial/mesenchymal states in 11 canine cancer cell lines derived from mammary gland, squamous cell carcinoma, lung, and melanoma. Sensitivity to atorvastatin varied among canine cancer cells, with IC50 values ranging from 5.92 to 71.5 μM at 48 h, which were higher than the plasma concentrations clinically achieved with statin therapy. Atorvastatin preferentially attenuated the proliferation of mesenchymal‐like cells. In particular, highly statin‐sensitive cells were characterized by aberrant expression of the ZEB family of EMT‐inducing transcription factors. However, ZEB2 silencing in highly sensitive cells did not induce resistance to atorvastatin. Taken together, these results suggest that high expression of ZEB is a characteristic of highly statin‐sensitive cells and could be a molecular marker for predicting whether cancers are sensitive to statins, though ZEB itself does not confer statin sensitivity.

Normalization is a crucial step in gene expression analysis to avoid misinterpretation. Reverse transcription-quantitative polymerase chain reaction was used to measure the expression of 10 candidate housekeeping genes in non-differentiated (ND) and differentiated (DI) 3T3-L1 cells on days 5 and 10. We used geNorm, NormFinder, BestKeeper, RefFinder, and the ∆Ct method to evaluate expression stability. The findings revealed that (1) the expression levels of the reference genes changed over time, even in non-differentiating cells, and (2) peptidylprolyl isomerase A ( Ppia ) and TATA box-binding protein ( Tbp ) were stable reference genes for 10 days in both undifferentiated and differentiated 3T3-L1 cells. Notably, the expression of known reference genes in non-differentiating cells was altered throughout the experiment.

Myogenesis, the formation of muscle fibers, is affected by certain glycoproteins, including chondroitin sulfate (CS), which are involved in various cellular processes. We aimed to investigate the mechanism underlying CS-E-induced suppression of myotube formation using the myoblast cell line C2C12. Differentiated cells treated with 0.1 mg/ml CS-E for nine days showed multinucleated and rounded myotubes with myosin heavy chain positivity. No difference was found between the CS-E-treated group with rounded myotubes and CS (−) controls with elongated myotubes in the levels of phospho-cofilin, a protein involved in the dynamics of actin cytoskeleton. Interestingly, N-cadherin, which is involved in the gene expression of myoblast fusion factors (myomaker and myomixer), was significantly downregulated at both the mRNA and protein levels following CS-E treatment. These results suggest that N-cadherin downregulation is one of the mechanisms underlying the CS-E-induced suppression of myotube formation.

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