[Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells].

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 µmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/ß-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, ß-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3ß, ß-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3ß protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, ß-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/ß-catenin signaling pathway.

Optimization of Ultrasonic Flavonoid Extraction from Saussurea involucrate, and the Ability of Flavonoids to Block Melanin Deposition in Human Melanocytes.

(1) Background: Flavonoids are the primary medicinal ingredient of Saussurea involucrate, which have significant antioxidant capacity. Optimizing the extraction of Saussurea involucrate flavonoids (SIFs) and exploring the ability to block melanin deposition caused by reactive oxygen can greatly promote the development of S. involucrate whitening products. (2) Methods: Ultrasonic extraction process was optimized using the Box-Behnken design (BBD) and response surface methodology (RSM). Then, the effect of SIFs on antioxidant activity and anti-deposition of melanin, and genes related to the melanin synthesis are studied. (3) Results: The optimal extraction procedures are as follows: the extraction time, ethanol content, and solvent ratio (v/w) are 64 min, 54%, and 54:1, respectively. The reducing activity and scavenging rates of 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical, and ABTS+ were promoted as more S. involucrate flavonoid extract was added. The SIFs extract induced a decrease in the melanin synthesis by inhibiting the human melanoma A375 cell tyrosinase activity. SIFs also depress expression of melanin synthesis related genes. (4) Conclusions: the highest SIFs content was obtained by using 54% ethanol and 54:1 solvent ratio (v/w) for 64 min. The extract of SIFs exhibited good ability of antioxidant and anti-deposition of melanin in human melanocytes.

Quinalizarin induces cycle arrest and apoptosis via reactive oxygen species-mediated signaling pathways in human melanoma A375 cells.

Quinalizarin, a bioactive and highly selective compound, is known to promote apoptosis in colon and lung cancer cells. However, studies evaluating quinalizarin-induced apoptosis in melanoma cells have not been conducted. In the present study, we investigated the underlying mechanisms of antimelanoma activity of quinalizarin in human melanoma A375 cells. The MTT assay and Trypan blue staining were used to evaluate the cell viability. The flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Western blot was used to detect the expression of cell cycle and apoptosis-related proteins, MAPK, and STAT3. The results revealed a significant dose and time dependent effect of quinalizarin on inhibiting proliferation in three kinds of human melanoma cells, and had no significant toxic effects on normal cells. Moreover, quinalizarin triggered G2/M phase cell arrest by modulating the protein expression levels of CDK 1/2, cyclin A, cyclin B, p21 and p27, and induced apoptosis by down-regulating the antiapoptotic protein Bcl-2 and upregulating the proapoptotic protein BAD, leading to the activation of caspase-3 and PARP in the caspase cascade in A375 cells. Quinalizarin treatment led to apoptosis of A375 cells via activation of MAPK and inhibition of STAT3 signaling pathways. In addition, quinalizarin increased the level of ROS, but ROS scavenger NAC inhibited quinalizarin-induced apoptosis by regulating MAPK and STAT3 signaling pathways. In summary, quinalizarin induces cell cycle arrest and apoptosis via ROS-mediated MAPK and STAT3 signaling pathways in human melanoma A375 cells, and quinalizarin may be used as a novel and effective antimelanoma therapeutic.

Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells / 中国中药杂志

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.

Effects and mechanism of oridonin on invasion and migration abilities of human melanoma A375 cells / 中草药

Objective To study the effects and the mechanism of oridonin on inhibiting the invasion and migration abilities of human melanoma A375 cells. Methods Human melanoma A375 cells were cultured and treated respectively with indicated concentrations of oridonin by cell culture technique. The proliferation rate was detected by CCK-8 method. The migration ability was measured by wound healing assay. The invasion ability was examined by Transwell assay. The adhesion capabilities were evaluated by adhesion assay. The epithelial-mesenchymal transition (EMT) and matrix metalloproteinases (MMPs) related protein expression levels were determined by Western blotting. Results CCK-8 assay showed the median inhibition concentration (IC50) of 48 h was 47.94 μmol/L. Oridonin (5, 10, and 20 μmol/L) inhibited the migration, invasion and adhesion abilities of human melanoma A375 cells in a dose-dependent manner (P < 0.05). After oridonin treatment, the protein expression levels of E-cadherin increased significantly (P < 0.05) and the protein levels of Snail, N-cadherin, vimentin, MMP-2, and MMP-9 decreased significantly (P < 0.05). Conclusion Oridonin inhibits the migration, invasion and adhesion abilities of human melanoma A375 cells. The mechanism may be related with the regulating effects of oridonin on EMT and MMPs.

Study on the Effect and Mechanism of Physcion 8-O-β-glucopyranoside on the Apoptosis of Skin Melano-ma A375 Cells / 中国药房

OBJECTIVE:To study the effect and mechanism of physcion 8-O-β-glucopyranoside(PG)on the apoptosis of skin melanoma A375 cells. METHODS:After A375 cells were treated by PG with 0,10,20,50 μg/mL for 24,48,72 h,CCK-8 method was adopted to determine the survival rate of cells. After A375 cells were treated by PG with 0(control),20,50 μg/mL for 48 h,flow cytometry was used to detect the apoptosis rate of cells with membrane protein Ⅴ/propidium iodide (PI) double staining. Immunoblotting was used to detect the protein expressions of Caspase-3 and polyadenyl adenine diphosphate ribose poly-merase (PARP) and protein expressions of cytochrome C inside and outside mitochondria. After A375 cells were treated by PG with 0 (control),5,10 μmol/L for 48 h,enzyme substrate method was used to determine the activities of Caspase-8 and Cas-pase-9. RESULTS:PG can effectively decrease the survival rate of A375 cells. Compared with control,apoptosis rate of cells was obviously increased after treated by PG with 20,50 μg/mL(P<0.01);protein expressions of Caspase-3,PARP in cells and cyto-chrome C in cell matrix were obviously enhanced(P<0.05 or P<0.01);and protein expression of cytochrome C in mitochondria was obviously weakened(P<0.05 or P<0.01). Caspase-9 activity in cells was obviously enhanced after treated by PG with 5,10μmol/L(P<0.05 or P<0.01);and Caspase-8 activity had no obvious changes. CONCLUSIONS:PG can inhibit activity of A375 cells and promote its apoptosis,and its pro-apoptotic effects is achieved by destructing mitochondrial membrane potential and pro-moting cytochrome C outflow.

Study on the Effect and Mechanism of Physcion 8-O-β-glucopyranoside on the Apoptosis of Skin Melano-ma A375 Cells / 中国药房

OBJECTIVE:To study the effect and mechanism of physcion 8-O-β-glucopyranoside(PG)on the apoptosis of skin melanoma A375 cells. METHODS:After A375 cells were treated by PG with 0,10,20,50 μg/mL for 24,48,72 h,CCK-8 method was adopted to determine the survival rate of cells. After A375 cells were treated by PG with 0(control),20,50 μg/mL for 48 h,flow cytometry was used to detect the apoptosis rate of cells with membrane protein Ⅴ/propidium iodide (PI) double staining. Immunoblotting was used to detect the protein expressions of Caspase-3 and polyadenyl adenine diphosphate ribose poly-merase (PARP) and protein expressions of cytochrome C inside and outside mitochondria. After A375 cells were treated by PG with 0 (control),5,10 μmol/L for 48 h,enzyme substrate method was used to determine the activities of Caspase-8 and Cas-pase-9. RESULTS:PG can effectively decrease the survival rate of A375 cells. Compared with control,apoptosis rate of cells was obviously increased after treated by PG with 20,50 μg/mL(P<0.01);protein expressions of Caspase-3,PARP in cells and cyto-chrome C in cell matrix were obviously enhanced(P<0.05 or P<0.01);and protein expression of cytochrome C in mitochondria was obviously weakened(P<0.05 or P<0.01). Caspase-9 activity in cells was obviously enhanced after treated by PG with 5,10μmol/L(P<0.05 or P<0.01);and Caspase-8 activity had no obvious changes. CONCLUSIONS:PG can inhibit activity of A375 cells and promote its apoptosis,and its pro-apoptotic effects is achieved by destructing mitochondrial membrane potential and pro-moting cytochrome C outflow.

Inhibitory effect of galangin on polyphenol oxidase and study of mechanism / 中国免疫学杂志

Objective:To investigated the activity inhibition and inhibitory type of polyphenol oxidase (PPO) induced by galangin and the interaction mechanism of galangin with polyphenol oxidase was preliminarily indicated,and prove the related mechanism of galangin on proliferation of on human melanoma A375 cells.Methods: The activity inhibition and inhibitory type of PPO induced by galangin were investigated by spectrophotometric method,and interation mechanism of galangin with PPO was preliminarily indicated by fluorescence quenching and molecular docking,and chelating copper ions with the inhibitory mechanism of galangin on polyphenol oxidase was measured.Results: Galangin was a competitive inhibitor,the IC50 and Ki on PPO were obtained to be (47.86±3.33) and (24.83±1.45)μmol/L,respectively.Fluorescence spectrum indicated the fluorescence of PPO was quenched effectively by galangin and the binding constant Ka was obtained to be (4.67±0.43)×104 L/mol.Chelating copper ions and molecular simulation further showed that galangin was combined with active center of copper ions,and formed hydrogen bonds with catalytic site His259.Luteolin could induce the apoptosis of A375 cells significantly,and the tyrosinase activity and melanin synthesis were decreased.Conclusion: Galangin as a competitive polyphenol oxidase inhibitor and reduced the activity of polyphenol oxidase.which provides the theoretical basis for the clinical anti skin cancer.