Preparation and in vitro evaluation of pH-sensitive charge-reversal nanoparticles loaded with curcumin / 中草药

Objective: To prepare charge-reversal pH-sensitive nanoparticles loaded with curcumin (PCE/Cur NPs), and investigate the optimizing technology, physicochemical characterizations, and inhibitory effect on B16 cell. Methods: The β-carboxyl amidized cationic MPEG-PCL-PEI polymers (PCE) were negatively charged, which were prepared into the negative PCE/Cur NPs with pH dependence. When pH > 7, there was no charge-reversal. When pH < 6, the β-carboxyl amides were hydrolyzed rapidly into corresponding amines. As a result, PCE/Cur NPs became positively charged again. The obtained PCE/Cur NPs were characterized by detection of particle size, morphology study, drug loading, encapsulation efficiency, and release study. The effect of anti-migratory and anti-invasive actions of PCE/Cur NPs on B16 cell was investigated using MTT assays and wound healing test. Results: PCE/Cur NPs dependent on pH charge inversion were successfully prepared. The obtained PCE/Cur NPs were round, and the size was uniform, the adhesion was not found. The Results: showed that the prepared PCE/Cur NPs had the highest DL (8.0 ± 1.0)%, EE (90.0 ± 2.0)%, mean particle size of (80 ± 5) nm, and zeta potential of (-35 ± 5) mV. Within 48 h, the accumulative release rate was (69.2 ± 5.2)% (pH 7.4) and (71.2 ± 4.3)% (pH 5), respectively, and then PCE/Cur NPs released slowly. These Results: by MTT assay and wound healing assay indicated that PCE/Cur NPs not only inhibited the proliferation of B16 cells in a concentration- and time-dependent manner, but also can induce apoptosis. Conclusion: PCE/Cur NPs were prepared successfully, which might have great potential application in drug delivery system.

Effect of polyphenol from Cortex Mori on melanogenesis of B16 cells and its mechanism / 中国药理学通报

Aim To investigate the inhibitory effect of polyphenol from Cortex Mori( CMP) on melanogenesis in mouse melanoma B16 cells and its possible mecha- nism. Methods Melanoma B16 cells with high ex-pression melanin were induced by α-melanocyte-stimu-lating hormone ( α-MSH) to establish cell model. Cell viability was detected by MTT assay. The melanin syn-thesis and tyrosinase activity were measured by NaOH and L-Dopa assays, respectively. The tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), tyrosi-nase-related protein-2 ( TRP-2 ) and microphthalmia associated transcription factor ( MITF ) protein and mRNA levels were measured by Western blot and qRT-PCR, respectively. Results CMP could inhibit the melanin synthesis and tyrosinase activity in α-MSH stimulated B16 cells in a dose-dependent manner ( P<0.05) . The melanin content and tyrosinase activity significantly decreased by 52.95% , 32.85% at 20 mg ·L-1of CMP, respectively. Treatment of 100 mg· L-1of arbutin reduced the melanin content and tyrosi- nase activity by 17.29% , 16.75% , respectively. Based on the results of this study, CMP showed a stronger anti-melanogenesis activity than that of positive control arbutin. After treated by CMP, the protein and mRNA levels of TYR, TRP-1, TRP-2 and MITF were significantly inhibited compared to the α-MSH group ( P<0.05) . Conclusions CMP could suppress the melanogenesis in α-MSH stimulated B16 cells, and its mechanism may be related to its regulation of the pro-tein and mRNA expressions of TYR, TRP-1, TRP-2 and MITF, and the inhibition of tyrosinase activity.

Cuprous oxide nanoparticles effect on the epithelial-mesenchymal transition of B16 cells / 药学实践杂志

Objective To research the epithelial-mesenchymal transition (EMT) effects of cuprous oxide nanoparticles (CONPs) on melanoma.Methods Cuprous oxide nanoparticles were prepared hydrothermally.The B16 cells were cultured with cuprous oxide nanoparticlesat different concentrations (5,25,50 μg/ml).The changes of the morphology of the B16 cell were observed under the inverted microscope.The effects of CONPs on B16 cell migration ability were detected through the Wound healing assay and the Transwell assay.Then cell immunofluorescence and western blotting were used to test the EMT related molecular markers, including E-cadherin, N-cadherin, Cytokeratin, and Vimentin.Results The synthesized cuprous oxide nanoparticles distribute uniformly with a diameter of 40 nm.Our study indicated that CONPs inhibited the EMT of B16 cell.A conversion process was discovered in this study.In B16 cells, CONPs inhibited B16 cell migration, promoted the expression of E-cadherin, Cytokeratin and Desmoplakin, while the expression of N-cadherin and Vimentin was repressed in protein level.Conclusion Cuprous oxide nanoparticles can significantly restrain the invasion and metastasis of melanoma cells and inhabit the EMT of B16 cells.

Inhibitory effect of p-hydroxylcinnamaldehyde from Momordicae Semen on growth of mouse melanoma xenograft in vivo andstudy on its mechanism / 中草药

Objective: To investigate the inhibitory effect of p-hydroxylcinnamaldehyde (PHD) from Momordicae Semen seeds on growth of mouse melanoma B16 cells in vivo. Methods: The inhibitory effect of PHD on growth of mouse melanoma B16 cells was measured by MTS method. Morphological changes of B16 cells were observed by phase contrast microscope. The xenograft tumor models of B16 cells in mice were established and divided into two groups: The mice in treatment group were treated with PHD (2 mg/kg) and in control group (treated with equal volume of normal saline). The growth of xenograft tumors in mice was observed and their sizes and weights were measured. The expression of Tyr, MMP-9, S-100B, p-P38, and p-ERK in tumor tissues was detected by immunohistochemical method. Morphological changes of lung and liver tissues in mice were observed by HE staining. Results: PHD had obvious inhibitory effect on the growth of B16 cells in vitro (P < 0.01). The morphological changes of B16 cells were typically differentiated after treated with 20 μmol/L PHD for 48 h. The average volume and weight of tumor tissues in mice of PHD treatment group were significantly decreased as compared with those of the control group (P < 0.01). Compared to the control group, the expression levels of Tyr and p-P38 in PHD treatment group were increased (P < 0.05), while the expression levels of MMP-9, S-100B, and p-ERK were decreased (P < 0.05). No obvious morphological changes were found in liver and lung tissues of mice in PHD treatment group and the control group. However, lung tumor metastasis was found in control group mice. Conclusion: PHD has inhibitory effect on the growth of xenograft tumor of mouse melanoma cells in mice.

Study on the Whitening activity and pathway of red eucommia herbs / 中国生化药物杂志

Objective To determine the impact of PEE with different ethanol concentration extract on the activity of tyrosinase in B16 melanoma and to explore the feasibility of PEE used as skin whitening agent make use of a model organism .Methods 30%, 50%, 70%, 95%, different concentration of ethanol reflux got four kinds of PEE alcohol extract, respectively.And selected the most active inhibition to mushroom tyrosinase ( PEE30、PEE50、PEE70、PEE95).The effect of extracts from the selected on B16 cell model was determined by MTS, NaOH splitting decomposition, L-DOPA oxidation progress.The change in protein expression level after ethanol extract of PEE was determined by Western bolt.Results The capacity of inhibition to mushroom tyrosinase in each concentration of PEE extraction, but PEE70 had the most active inhibition.When challenged with B16 cell model, PEE70 showed the capacity of decrease in tyrosinase activities and melanin synthesis (P<0.01) and had a does dependence ;it also decreased Tyrosinase and TRP-1 expression compared with control B16 cells.Conclusion These data support the idea that PEE can restrain melanogenesis, through its inhibitory effect on the activity of tyrosinase.

Effect of p-hydroxylcinnamaldehyde on differentiation of melanoma cells and its mechanism / 中草药

Objective: To explore the monomer compounds in the seeds of Momrodica cochinchinensis and to study the differentiation of mouse melanoma B16 cells induced by p-hydroxylcinnamaldehyde (PHC). Methods: After being treated by five kinds of compounds [PHC, coniferylaldehyde, p-hydroxylbenzaldehyde (PHB), 3-O-methoxyaniline-p-hydroxylbenzaldehyde, and ligballinol] for 48 h, the inhibitory rate of B16 cell growth was measured by sulforhadamine B (SRB); Morphological changes of B16 cells induced by PHC for 24, 48, and 72 h were observed by Giemsa staining and phase contrast microscope; Melanin content and the activity of tyrosinase in B16 cells 48 h after the administration were assessed by colorimeter. The expression of tyrosinase mRNA was detected by RT-PCR. Results: All the five compounds had the inhibitory effect on the B16 cells. Among them, PHC showed the strongest effect in the dose-and time-dependent manner; PHC could induce B16 cells dendritic growth 48 h after the treatment, and the morphological changes were typically differentiated; PHC also increased the melanin production and the activity of tyrosinase. There was a significant difference compared to the control group (P < 0.05). After treated by PHC for 6, 12, and 24 h, the expression levels of tyrosinase mRNA, tyrosinase 1 mRNA, and tyrosinase 2 mRNA were significantly increased (P < 0.01) in a time-dependent manner. Conclusion: PHC could inhibit the proliferation of B16 cells and the mechanism is related to the differentiation of B16 cells.

Autophagic cell death in mouse melanoma B16 cells induced by TNF-α / 国际生物医学工程杂志

ObjectiveTo analyze the autophagy of mouse melanoma B16 cells induced by TNF-ct.MethodsMouse melanoma B 16 cells treated with TNF-α were used in this study.The expression levels of LC3- Ⅱand Beclinl were measured by western blot and mRNA levels of autophagy related gene atg5,atg7 and atgl2 were measured by reverse transcription polymerase chain reaction (RT-PCR) after TNF-α treatment.ResultsThe viability of B16 cells were obviously inhibited after incubation with TNF-α.The expression levels of autophagy related protein LC3- Ⅱ and Beclinl were elevated in TNF-α treated B16 cells compared with in control B16 cells.The mRNA expression levels of autophagy related gene Atg5,atg7 and atg12 showed consistent up regulation in TNF-a treated B16 cells compared with in control B16 cells.ConclusionTNF-α can induce autophagic cell death in B16 cells.

The regional genomic instability induced by 60Co γ-rays in B16 cells transfected by GFP / 中华放射医学与防护杂志

Objective To detect the regional genomic instability of B16 cells treated with 60Co γ-rays by a green fluorescence protein (GFP)-based genomic instability reporting system.Methods Three groups were employed as non-transfection group,vector control group and transfection group.The GFP-marked reporter construct pCMV-EGFP2XhoI for regional genomic instability was successfully transfected into B16 cells using liposome.B16 cells were selected by screening of G418 with a series of concentrations and limiting dilution cultures to yield a single colony.B16 cells with the genomic instability report system were then irradiated by 60Co γ-rays at doses of 0,2 and 4 Gy.The regional genomic instability of B16 cellswas quantified by counting the number of cells with GFP expression.Results B-16 cell strain steadilyexpressing the GFP-based genomic instability reporting system was established successfully.GFP-positiveB16 cells were observed at 1 d after irradiation with 60Co γ-rays at doses of 2 and 4 Gy.Positive correlations between fluorescence intensity and dose and fluorescence intensity and time were also observed.The positive expression rate of GFP followed the increased of dose (F =36.55,36.76,P ＜ 0.05) and time (t =-3.27,-3.16,-4.26,-6.11,-7.17,P ＜ 0.05),and differences between groups were significant.The positive expression rate of GFP increased significantly at 3 d,and maximum expression was observed at 5 d(2.46 ± 0.24 and 3.82 ± 0.35).The level was tending towards stability.Spontaneous GFP expression at a ratio of 1/600 000 was observed in 0 Gy group after 2 weeks of culture.Conclusions The regional genomic instability of B16 cells induced by 60Co γ-rays can be detected using a GFP-labelled genomic instability reporter system.

Screening of conditions carrying murine melanoma B16 cells in spaceflights without care / 中国康复理论与实践

@#Objective To screen the optimum conditions carrying murine melanoma B16 cells in spaceflights without cares.MethodsMurine melanoma cells were cultured on micocarriers and grouped depending on cells concentration, serum concentration, microcarrier number and temperature.After 33 days, B16 cells were stained by Giemsa, observed with phase-contrast microscope and counted for surviving percentage.ResultsThe optimum conditions,in which the surviving percentages were 8% and 10%, were obtained in the experiments.B16 cells were carried in the 20th recoverable satellite orbiting 18 days under the optimum conditions. After recovering, 110 strain monocloned cells were survived and the surviving percentage was 1.1%.ConclusionThe optimum conditions carrying murine melanoma B16 cells in spaceflights without cares seems to be obtained,and it did improve the surviving time and percentage of cells in spaceflights without cares.

Trichosanthin-induced apoptosis in melanoma B-16 cell line / 中国病理生理杂志

AIM: To investigate the antitumor effect and mechanism of trichosanthin (TCS) on melanoma B-16 cells. METHODS: (1) The injury of B-16 cells by trichosanthin was observed with SCGE and hoechst33258 staining method. (2) LCSM and specificity fluorescent probe Fluo-3/AM, H2DCF-DA, DAF-FM diacetate were applied to analyze the dynamic changes of Ca2+, ROS and NO in single cell cultured with TCS. Simultaneously, the relationship between ROS, NO and increase of Ca2+ was also revealed. RESULTS: (1)When treated with TCS (50 mg/L) for 3 h and 6 h, neither cytotoxicity assay nor SCGE showed the differences compared with control group. After 12 h incubation, specificity phenomena of DNA injury-comet tail appeared in SCGE and chromatin condensation even apoptotic body formation were seen by Hoechst33258 staining. (2) TCS (50 mg/L) evoked rapid enhancement of the production of Ca2+, ROS and NO in the cell and the differences between TCS and control group had statistical significance (P