Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion

The cholesterol reducing drugs, statins, exhibit anti-tumor effects against cancer stem cells and various cancer cell lines, exert potent additivity or synergy with existing chemotherapeutics in animal models of cancer and may reduce cancer incidence and cancer related mortality in humans. However, not all tumor cell lines are sensitive to statins, and clinical trials have demonstrated mixed outcomes regarding statins as anticancer agents. Here, we show that statin-induced reduction in intracellular cholesterol levels correlate with the growth inhibition of cancer cell lines upon statin treatment. Moreover, statin sensitivity segregates with abundant cytosolic vimentin expression and absent cell surface E-cadherin expression, a pattern characteristic of mesenchymal-like cells. Exogenous expression of cell surface E-cadherin converts statin- sensitive cells to a partially resistant state implying that statin resistance is in part dependent on the tumor cells attaining an epithelial phenotype. As metastasizing tumor cells undergo epithelial to mesenchymal transition during the initiation of the metastatic cascade, statin therapy may represent an effective approach to targeting the cells most likely to disseminate

Jedinstveni obrazac djelovanja bisfenola A na ekspresiju gena čimbenika rasta živca embrionske mišje stanične linije N-44 dobivene iz hipotalamusa

We investigated the toxicity of bisphenol A (BPA) by determining the gene expression of nerve growth factor (Ngf) in the embryonic mouse cell line mHypoE-N44 derived from the hypothalamus exposed to BPA dose range between 0.02 and 200 μmol L-1 for 3 h. Ngf mRNA levels decreased in a dose-dependent manner, with significant reductions observed in the 2 to 50 μmol L-1 BPA treatment groups compared to controls. However, at 100 to 200 μmol L-1 the Ngf mRNA gradually increased and was significantly higher than control, while the expression of the apoptosis-related genes Caspase 3 and transformation-related protein 73 decreased significantly. These results suggest that in an embryonic hypothalamic cell line the higher doses of BPA induce a unique pattern of Ngf gene expression and that BPA has the potential to suppress apoptosis essential for early-stage brain development.U istraživanju toksičnosti bisfenola A (BPA) utvrđena je ekspresija gena čimbenika rasta živca (eng. nerve growth factor - NGF) embrionske mišje stanične linije mHypoE-N44 dobivene iz hipotalamusa nakon trosatnog izlaganja BPA-u u rasponu doza od 0,02 do 200 μmol L-1. Razine Ngf mRNA snizile su se ovisno o dozi, a značajne razlike od kontrolne skupine zamijećene su za raspon od 2 do 50 μmol L-1. Međutim, počevši od doze od 100 do 200 μmol L-1, razine Ngf mRNA značajno su se povećale u odnosu na kontrolu, a ekspresija gena kaspaze 3 i transformacijskog proteina 73 značajno snizila. Ti rezultati upućuju na to da visoke doze BPA u embrionskoj hipotalamičkoj staničnoj liniji stvaraju jedinstveni obrazac ekspresije gena Ngf te da BPA može suprimirati apoptozu koja je nužna za rani razvoj mozga

Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C2C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion

Glycerol induces early fibrosis in regenerating rat skeletal muscle

Glycerol has been recently used to induce muscle adiposity in mice. However, its effects on the rat muscles have not been investigated previously. Therefore, we investigated the regeneration outcomes of rat muscles following glycerol-induced injury at different time points. Glycerol injection induced myofiber degeneration with extensive inflammatory infiltration on day 4 followed by appearance of regenerating myotubes on day 7 after injury without adipocyte infiltration. Meanwhile, a significant collagen deposition at early stage of regeneration that increased together with persistent inflammatory infiltration up to day 14 after injury indicates impaired regeneration. In conclusion, glycerol injury in rats is more suitable as a fibrosis-inducing model than in mice due to earlier and higher accumulation of fibrous tissue with lacking adipogenesis.JSPS KAKENHI Grant Number 16H02585 and the Egyptian Government.https://www.jstage.jst.go.jpam2019Anatomy and Physiolog

Comparison of Characterization in Two-Dimensional and Three-Dimensional Canine Mammary Gland Tumor Cell Models

Background: Canine mammary gland tumors can be used as predictive models for human breast cancer. There are several types of microRNAs common in human breast cancer and canine mammary gland tumors. The functions of microRNAs in canine mammary gland tumors are not well understood. Methods: We compared the characterization of microRNA expression in two-dimensional and three-dimensional canine mammary gland tumor cell models. We evaluated the differences between two- and three-dimensional cultured canine mammary gland tumor SNP cells by assessing microRNA expression levels, morphology, drug sensitivity, and hypoxia. Results: The expression of microRNA-210 in the three-dimensional-SNP cells was 10.19 times higher than that in the two-dimensional-SNP cells. The intracellular concentrations of doxorubicin in the two- and three-dimensional-SNP cells were 0.330 ± 0.013 and 0.290 ± 0.048 nM/mg protein, respectively. The IC50 values of doxorubicin for the two- and three-dimensional-SNP cells were 5.2 and 1.6 μM, respectively. Fluorescence of the hypoxia probe, LOX-1, was observed inside the sphere of three-dimensional-SNP cells without echinomycin but not in two-dimensional-SNP cells. The three-dimensional-SNP cells treated with echinomycin showed weak LOX-1 fluorescence. Conclusion: The present study showed a clear difference in microRNA expression levels in cells cultured in a two-dimensional adherent versus a three-dimensional spheroid model

CYP11A1 silencing suppresses HMGCR expression via cholesterol accumulation and sensitizes CRPC cell line DU-145 to atorvastatin

Statins, which are cholesterol synthesis inhibitors, are well-known therapeutics for dyslipidemia; however, some studies have anticipated their use as anticancer agents. However, epithelial cancer cells show strong resistance to statins through an increased expression of HMG-CoA reductase (HMGCR), an inhibitory target of statins. Castration-resistant prostate cancer (CRPC) cells synthesize androgens from cholesterol on their own. We performed suppression of CYP11A1, a rate-limiting enzyme in androgen synthesis from cholesterol, using siRNA or inhibitors, to examine the effect of steroidogenesis inhibition on statin sensitivity in CRPC cells. Here, we suggested that CYP11A1 silencing sensitized the statin-resistant CRPC cell line DU-145 to atorvastatin via HMGCR downregulation by an increase in intracellular free cholesterol. We further demonstrated that CYP11A1 silencing induced epithelial-mesenchymal transition, which converted DU-145 cells into a statin-sensitive phenotype. This suggests that concomitant use of CYP11A1 inhibitors could be an effective approach for overcoming statin resistance in CRPC. Moreover, we showed that ketoconazole, a CYP11A1 inhibitor, sensitized DU-145 cells to atorvastatin, although not all the molecular events observed in CYP11A1 silencing were reproducible. Although further studies are necessary to clarify the detailed mechanisms, ketoconazole may be effective as a concomitant drug that potentiates the anticancer effect of atorvastatin

Housekeeping gene expression variability in differentiating and non-differentiating 3T3-L1 cells

ABSTRACTNormalization 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

Degree of Suppression of Mouse Myoblast Cell Line C2C12 Differentiation Varies According to Chondroitin Sulfate Subtype

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C2C12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion