Celastrus orbiculatus Thunb. extract inhibits EMT and metastasis of gastric cancer by regulating actin cytoskeleton remodeling.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine herb Celastrus orbiculatus Thunb. is an important folk medicinal plant in China that has been used as an anti-inflammatory, antitumor, and analgesic in various diseases. Recent years, many studies have reported the significant effects of Celastrus orbiculatus Thunb. extract (COE) on gastric cancer. However, the specific mechanism by which COE regulates gastric cancer cytoskeleton remodeling and thus inhibits EMT has not yet been reported. AIM OF STUDY: To study the effect and mechanism of COE in inhibiting the epithelial-mesenchymal transition (EMT) and metastasis of gastric cancer cells, laying an experimental foundation for the clinical application and further development of COE. METHODS: The high-content cell dynamic tracking system was used to continuously track the trajectory of cell movement in real time. Through the high-content data, the average movement distance and movement speed of the cells are calculated. Additionally, the dynamic images of the cell movement in the high-content imaging system are derived to analyze the impact of COE on the movement of gastric cancer cells. Cytoskeleton staining experiment was performed to detect the effect of COE on the assembly of gastric cancer cell cytoskeleton proteins. Western blot was employed to detect the changes of EMT and metastasis-related proteins in the gastric cancer cells treated by COE. The effect of COE on the key regulatory protein Cofilin-1 (CFL1) of cell movement was examined by Western blot and protein degradation experiment. The effect of COE on EMT and metastasis of the gastric cancer cells lacking CFL1 was assessed by a transwell assay. The in vivo inhibitory effect of COE on EMT and metastasis of gastric cancer was determined by the animal living image system. IHC assays were used to detect the levels of EMT-related proteins in COE reversal in vivo. RESULT: The results showed that the movement distance and average movement speed of gastric cancer cells after COE treatment were significantly lower than those of the control group. Cytoskeleton staining experiments revealed that COE can significantly change the distribution of skeletal proteins in gastric cancer cells. Additionally, COE treatment significantly reduced the expression of Matrix metalloproteinases (MMP-2, MMP-9) and other proteins. Furthermore, COE can significantly accelerate the degradation of CFL1 protein, and both COE treatment and CFL1 deletion can significantly inhibit EMT and metastasis of gastric cancer cells. Lastly, the number of peritoneal metastases of gastric cancer cells was significantly reduced in animals after COE treatment. COE can reverse the levels of EMT-related proteins while reducing the expression levels of CFL1 protein in vivo. CONCLUSION: COE can significantly inhibit EMT and metastasis of gastric cancer cells in vivo and in vitro. This effect may be achieved by reducing the stability of CFL1 and inhibiting the assembly of actin in gastric cancer cells.

Brain ischemic insult induces cofilin rod formation leading to synaptic dysfunction in neurons.

Ischemic stroke not only induces neuron death in the infarct area but also structural and functional damage of the surviving neurons in the surrounding peri-infarct area. In the present study, we first identified cofilin rod, a pathological rod-like aggregation, formed in neurons of in vivo ischemic stroke animal model and induced neuronal impairment. Cofilin rods formed only on the ipsilateral side of the middle cerebral artery occlusion and reperfusion (MCAO-R) rat brain and showed the highest density in peri-infarct area. Our real-time live cell imaging, immunostaining and patch clamp studies showed that cofilin rod formation in neurons led to dendritic mitochondrial transportation failure, as well as impairment of synaptic structure and functions. Overexpression of LIM kinase or activation of its upstream regulator Rho, suppressed ischemia-induced cofilin rod formation and showed protective effect on synaptic function and structure impairment in both cultured neurons and MCAO-R rat model. In summary, our results demonstrate a novel mechanism of ischemic stroke-induced neuron injury in peri-infarct area and provide a potential target for the protection of neuronal structure and function against brain ischemia insult.

Angiotensin II type 2 receptor stimulation increases the rate of NG108-15 cell migration via actin depolymerization.

Angiotensin II (Ang II) has been reported to induce migration in neuronal cell types. Using time-lapse microscopy, we show here that Ang II induces acceleration in NG108-15 cell migration. This effect was antagonized by PD123319, a selective AT2 receptor antagonist, but not by DUP753, a selective AT1 receptor antagonist, and was mimicked by the specific AT2 receptor agonist CGP42112. This Ang II-induced acceleration was not sensitive to the inhibition of previously described signaling pathways of the AT2 receptor, guanylyl cyclase/cyclic GMP or p42/p44 mapk cascades, but was abolished by pertussis toxin treatment and involved PP2A activation. Immunofluorescence studies indicate that Ang II or CGP42112 decreased the amount of filamentous actin at the leading edge of the cells. This decrease was accompanied by a concomitant increase in globular actin levels. Regulation of actin turnover in actin-based motile systems is known to be mainly under the control of the actin depolymerizing factor and cofilin. Basal migration speed decreased by 77.2% in cofilin-1 small interfering RNA-transfected NG108-15 cells, along with suppression of the effect of Ang II. In addition, the Ang II-induced increase in cell velocity was abrogated in serum-free medium as well as by genistein or okadaic acid treatment in a serum-containing medium. Such results indicate that the AT2 receptor increases the migration speed of NG108-15 cells and involves a tyrosine kinase activity, followed by phosphatase activation, which may be of the PP2A type. Therefore, the present study identifies actin depolymerization and cofilin as new targets of AT2 receptor action, in the context of cellular migration.

Activity of cofilin can be regulated by a mechanism other than phosphorylation/dephosphorylation in muscle cells in culture.

Cofilin plays a critical role in actin filament dynamics in a variety of eukaryotic cells. Its activity is regulated by phosphorylation/dephosphorylation of a Ser3 residue on the N-terminal side and/or its binding to a phosphoinositide, PIP(2). To clarify how cofilin activity is regulated in muscle cells, we generated analogues of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser3) of cofilin to Ala and Asp, respectively. These mutated proteins, as well as the cofilin having Ser3 residue (S3-cofilin), were produced in an E. coli expression system and conjugated with fluorescent dyes. In an in vitro functional assay, A3-cofilin retained the ability to bind to F-actin. Upon injection into cultured muscle cells, A3-cofilin and S3-cofilin promptly disrupted actin filaments in the cytoplasm, and many cytoplasmic rods containing both the exogenous cofilin and actin were generated, while D3-cofilin was simply diffused in the cytoplasm without affecting actin filaments. Several hours after the injection, however, the activity of A3-cofilin and S3-cofilin was suppressed: the actin-A3-cofilin (or S3-cofilin) rods disappeared, the cofilin diffused in the cytoplasm like D3-cofilin, and actin filaments reformed. Both GFP-fused A3-cofilin and S3-cofilin that were produced by cDNA transfection were also suppressed in the cytoplasm of muscle cells in culture. Thus, some mechanism(s) other than phosphorylation can suppress A3-cofilin activity. We observed that PIP(2) can bind to A3-cofilin just as to S3-cofilin and inhibits the interaction of A3-cofilin with actin. Our results suggest that the activity of A3-cofilin and also S3-cofilin can be regulated by PIP(2) in the cytoplasm of muscle cells.