β-escin reverses multidrug resistance through inhibition of the GSK3β/β-catenin pathway in cholangiocarcinoma

AIM: To develop a safe and effective agent for cholangiocarcinoma(CCA) chemotherapy. METHODS: A drug combination experiment was conducted to determine the effects of β-escin in c o m b i n a t i o n w i t h c h e m o t h e ra p y o n C C A c e l l s. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of β-escin and common chemotherapeutics on the proliferation of human CCA cells(QBC939, Sk-Ch A-1, and MZ-Ch A-1). Immunocytochemistry was used to detect the expression of P-glycoprotein(P-gp) protein. Luciferase reporter assay was used to detect the activation of the Wnt/β-catenin pathway. The protein levels of P-gp, p S9-GSK3β, p T216-GSK3β, GSK3β, β-catenin, and p-β-catenin were further confirmed by western blotting.RESULTS: The drug sensitivity of QBC939 and QBC939/5-fluorouracil(5-FU) cells to 5-FU, vincristine sulfate(VCR), or mitomycin C was significantly enhanced by β-escin compared with either agent alone(P < 0.05). In addition, the combination of β-escin(20 μmol/L) with 5-FU and VCR was synergic with a combination index < 1. Further investigation found that the m RNA and protein expression of P-gp was downregulated by β-escin. Moreover, β-escin induced GSK3β phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of β-catenin. Interestingly, activation of the GSK3β/β-catenin pathway induced by Wnt3 a resulted in upregulation of P-gp, which was effectively abolished by β-escin, indicating that β-escin down-regulated P-gp expression in a GSK3β-dependent manner.CONCLUSION: β-escin was a potent reverser of P-gpdependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3β/β-catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA.

Apoptosis of human cholangiocarcinoma cells induced by ESC-3 from Crocodylus siamensis bile

AIM:To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells.METHODS:ESC-3 was isolated from crocodile bile by Sephadex LH-20 and RP-18 reversed-phase column.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was conducted to determine the effects of ESC-3 on the proliferation of human cholangiocarcinoma cell lines (QBC939,Sk-ChA-1 and MZ-ChA-1).Giemsa staining,Hoechst 33258 and acridine orange/ethidium bromide staining showed the morphological changes of Mz-ChA-1 cells exposed to ESC-3 at different concentrations.Flow cytometry with regular propidium iodide (PI) staining was performed to analyze the cell cycle distribution of Mz-ChA-1 cells and to assess apoptosis by annexin v-fluorescein isothiocyanate (VFITC)/PI staining.Rh123 staining was used to detect the alteration of mitochondrial membrane potential (ΔΨm).The protein levels of Bax,Bcl-2,Cdk2,cytochrome c and caspase-3 were further confirmed by Western blotting.RESULTS:ESC-3 significantly inhibited the growth of three human cholangiocarcinoma cell lines and arrested Mz-ChA-1 cell cycle at G0/G1 phase.Mz-ChA-1 cells showed typical apoptotic morphological changes after treated with ESC-3 (10 μg/mL) for 48 h.Cell death assay indicated that Mz-ChA-1 cells underwent apoptosis in a dose-dependent manner induced by ESC-3.In addition,ESC-3 treatment could downregulate the protein level of Bcl-2 and upregulate the Bax,leading to the increase in the ratio of Bax to Bcl-2 in Mz-ChA-1 cells.Meanwhile,cytochrome c was released from the mitochondria into the cytosol,which subsequently initiated the activation of caspase-3.All these events were associated with the collapse of the mitochondrial membrane potential.CONCLUSION:ESC-3,the active ingredient of crocodile bile,induced apoptosis in Mz-ChA-1 cells through the mitochondria-dependent pathway and may be a potential chemotherapeutic drug for the treatment of cholangiocarcinoma.

Enzymatic properties of phenoloxidase from Pieris rapae （Lepidoptera） larvae

The kinetic parameters of partially purified phenoloxidase （PO, EC. 1.14.18.1） from the 5th instar larvae of Pieris rapae （Lepidoptera） were determined, using L-3, 4- dihydroxyphenylalanine （L-DOPA） as substrate. The optimal pH and temperature of the enzyme for the oxidation of L-DOPA were determined to be at pH 7.0 and at 42℃, respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37℃. At pH 6.8 and 37℃, the Michaelis constant （Kin） and maximal velocity （V） of the enzyme for the oxidation of L-DOPA were determined to be 0.80 mmol/L and 1.84 μmol/ L/min, respectively. Tetra-hexylresorcinol and 4-dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC50 of 1.50 and 1.12 μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47μmol/L, respectively.

Optimization of extraction of phenolics from leaves of Ficus virens

In this research,the conditions for extraction of phenolics from leaves of Ficus virens were optimized using response surface methodology(RSM).The extraction abilities of phenolics(EAP) and flavonoids(EAF),the 2,2-diphenyl-1-pierylhydrazyl(DPPH) free-radical scavenging potential,and the ferric reducing/antioxidant power(FRAP) were used as quality indicators.The results of single-factor experiments showed that temperature,ethanol concentration,extraction time,and the number of extraction cycles were the main influencing variables,and these provided key information for the central composite design.The results of RSM fitted well to a second degree polynomial model and more than 98% of the variability was explained.The ideal extraction conditions for EAP,EAF,DPPH free-radical scavenging potential,and FRAP were obtained.Considering the four quality indicators overall,the ideal extraction conditions were 58% ethanol at 57 °C for 37 min with three extraction cycles.At the ideal extraction conditions,the values of EAP,EAF,DPPH free-radical scavenging potential,and FRAP were 5.72%,3.09%,58.88 mg ascorbic acid equivalent(AAE)/g dry weight(DW),and 15.86 mg AAE/g DW,respectively.In addition,linear correlations were observed between EAP,EAF,and antioxidant potential.