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Objective:To explore whether microRNA (miRNA) -181b-5p inhibits the proliferation and invasion of cutaneous melanoma cells by targeting pleckstrin (PLEK) .Methods:Bioinformatics methods were used to analyze cutaneous melanoma-associated core genes; dual-luciferase reporter assay was performed to verify the targeted interaction between miRNA-181b-5p and PLEK. Oligo RNA and small interfering RNA (siRNA) were used to regulate the expression of miRNA-181b-5p and PLEK in A375 cells respectively in this experiment, and A375 cells were divided into the following groups in detail: mimic negative control group, miRNA-181b-5p mimic group, inhibitor negative control group, miRNA-181b-5p inhibitor group, PLEK siRNA group, siRNA negative control group, miRNA-181b-5p inhibitor + control siRNA co-transfection group and miRNA-181b-5p inhibitor + PLEK siRNA3 co-transfection group. After 48-hour treatment, qPCR was performed to determine the mRNA expression of miRNA-181b-5p and PLEK in A375 cells, Western blot analysis to determine the PLEK protein expression, and Transwell assay to assess the invasive ability of A375 cells; after additional 24-96 hours of culture, cell counting kit-8 (CCK8) assay was conducted to assess the proliferative ability of A375 cells.Results:PLEK was the core gene for cutaneous melanoma. PLEK expression in the cutaneous melanoma in situ tissues was significantly higher than that in the paracancerous tissues ( P = 0.031) , but lower than that in the metastatic tissues ( P = 0.001) . Compared with human epidermal melanocytes HEMa-LP, the mRNA and protein expression of PLEK significantly increased in A375 cells (mRNA: 3.884 ± 0.156 vs. 0.997 ± 0.010, t = 18.48, P < 0.001; protein: 2.840 ± 0.301 vs. 1.029 ± 0.094, t = 5.47, P = 0.005) , but the miRNA-181b-5p expression significantly decreased in A375 cells (0.333 ± 0.042 vs. 0.967 ± 0.069, t = 7.83, P = 0.001) . Dual-luciferase reporter assay showed targeted binding of miRNA-181b-5p to PLEK. Compared with the mimic negative control group, the miRNA-181b-5p mimic group showed significantly decreased survival rate of A375 cells (48 hours: t = 7.96, P = 0.015; 72 hours: t = 7.50, P = 0.002; 96 hours: t = 7.96, P = 0.001) , and significantly decreased invasive ability of A375 cells ( t = 5.07, P = 0.007) ; on the contrary, the survival rate and invasive ability of A375 cells were significantly higher in the miRNA-181b-5p inhibitor group than in the inhibitor negative control group (survival rate: 24 hours, t =5.38, P = 0.013; 48 hours, t = 5.36, P = 0.013; 72 hours, t =7.63, P = 0.005; 96 hours, t = 5.99, P = 0.004; invasive ability: t = 7.24, P = 0.002) ; compared with the siRNA negative control group, the proliferative and invasive ability of A375 cells significantly decreased in the PLEK siRNA group (proliferative ability: 48, 72, 96 hours, P = 0.015, 0.011, 0.001, respectively; invasive ability: t = 4.93, P = 0.008) ; compared with the miRNA-181b-5p inhibitor + control siRNA co-transfection group, the miRNA-181b-5p inhibitor + PLEK siRNA co-transfection group showed significantly decreased proliferation rate and invasive ability of A375 cells (proliferation rate: 24, 48, 72, 96 hours, P = 0.042, 0.042, 0.037, 0.017, respectively; invasive ability: t = 8.52, P = 0.001) . Conclusion:miRNA-181b-5p can inhibit the proliferation and invasion of cutaneous melanoma A375 cells, likely by down-regulating the PLEK expression.
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Objective@#To evaluate the effect of overexpression of the autophagy marker gene Beclin1 on biological behaviors of SK-MEL-2 human malignant melanoma cells.@*Methods@#Western blot analysis was performed to determine the protein expression of Beclin1 in melanoma cell lines A375 and SK-MEL-2. SK-MEL-2 cells with low Beclin1 protein expression were selected as research objects, and divided into 3 groups: blank group receiving no treatment, negative control group transfected with pcDNA.3.1/myc-His (-) A, and experimental group transfected with pcDNA3.1-Beclin1 plasmid. After 2-week culture, cell counting kit-8 (CCK-8) assay was conducted to evaluate the effect of Beclin1 on cell proliferation at 24, 48 and 72 hours, and Transwell assay and wound-healing assay were performed to assess the effect of Beclin1 overexpression on the invasion and migration abilities of SK-MEL-2 cells. Repeated measures analysis of variance and completely randomized analysis of variance were used to analyze differences in indices among groups, and least significant difference (LSD) -t test was used for multiple comparisons.@*Results@#The protein expression of Beclin1 was significantly lower in the SK-MEL-2 cells (0.037 ± 0.010) than in the A375 cells (0.670 ± 0.150, F = 46.62, P<0.05) . The experimental group showed significantly increased protein expression of Beclin1 (0.32 ± 0.04) compared with the negative control group (0.06 ± 0.02, P < 0.05) and blank group (0.07 ± 0.02, P < 0.05) . CCK-8 assay revealed a significant difference in the cell proliferation rate among different groups and different time points (F = 1 077.36, 4 903.04 respectively, both P<0.05) , and there was a significant interaction between the transfection treatment and time (F= 205.20, P<0.05) . Transwell assay showed that the number of SK-MEL-2 cells crossing the chamber per high-power field (× 200) after 24-hour treatment was significantly lower in the experimental group (18.67 ± 1.19) than in the negative control group (87.89 ± 6.05, P<0.05) and blank group (86.78 ± 5.93, P<0.05) . In the wound-healing assay, the cell migration distance was significantly shorter in the experimental group than in the blank group and negative control group at 24 and 48 hours (all P < 0.05) .@*Conclusion@#Beclin1 overexpression can markedly inhibit the proliferation, invasion and migration of SK-MEL-2 cells.
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Objective To evaluate the effect of nucleolar protein 14 (NOP14) on angiogenesis in melanoma.Methods Melanoma tissues were collected from 40 patients with pathologically diagnosed melanoma in Guangzhou First People's Hospital from January 2016 to December 2018,and immunohistochemical study was conducted to determine the expression of NOP14 and CD31 (expressed as microvessel density [MVD]).Melanoma cell lines A375 and SK-MEL-1 were both divided into 4 groups:empty vector group transfected with the empty vector,NOPI4 group transfected with a NOP14-overexpressing vector,siNOP14 group transfected with the siRNA targeting NOP14,and siNC group transfected with a negative control siRNA.Fluorescence-based quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of NOP14 respectively,and Western blot analysis and enzyme-linked immunosorbent assay (ELISA) to measure the expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) in cells and their culture media.Coculture models of human umbilical vein endothelial cells (HUVECs) and A375/SK-MEL-1 cells in the above groups were established in Transwell chambers,and cell counting kit-8 (CCK8) assay,Transwell migration and invasion assays and Matrigel-based vasculogenic mimicry assay were performed to evaluate the cellular proliferative,migratory,invasive activity and tube formation capacity respectively.A linear regression model was used to analyze the relationship between NOP14 expression and MVD in melanoma tissues,multi-way analysis of variance to analyze the difference in cellular proliferative activity,and independent-sample t test to compare other experimental indices between 2 groups.Results The expression of CD31 (MVD) was 44 ± 13 in the group with high NOP14 expression (n =20),58 ± 16 in that with moderate NOP14 expression (n =17),and 62 ± 11 in that with low NOP14 expression (n =3).The NOP14 expression was negatively correlated with MVD (r =-0.525,P =0.017).Compared with the empty vector group,the expression of VEGF and VEGFR in A375 and SK-MEL-1 cells and their culture media significantly decreased in the NOP14 group (all P ＜ 0.05).Compared with the siNC group,the expression of VEGF and VEGFR in the A375 and SK-MEL-1 cells and their culture media significantly increased in the siNOP14 group(all P ＜ 0.05).In the co-culture models of A375 cells and HUVECs,the NOP14 group showed significantly decreased proliferative activity of HUVECs (F =131.85,P ＜ 0.05),and numbers of migratory cells (22 ± 5 vs.63 ± 8,t =7.07,P =0.002),invasive cells (14 ± 5 vs.45 ± 10,t =4.94,P =0.008) and branch points (8 ± 2 vs.14 ± 3,t =5.06,P ＜ 0.001) compared with the empty vector group;compared with the siNC group,the siNOP14 group showed significantly increased proliferative activity of HUVECs (F =79.92,P ＜ 0.01),and numbers of migratory cells (152 ± 30 vs.59 ± 4,t =5.36,P =0.006),invasive cells (134 ± 21 vs.50 ± 8,t =6.40,P ＜ 0.001) and branch points (27 ± 3 vs.15 ± 4,t =6.10,P ＜ 0.001).In the co-culture models of SK-MEL-1 cells and HUVECs,the 4 groups showed the same trend of changes in the cellular proliferative,migratory,invasive activity and tube formation capacity of HUVECs as the above groups in the co-culture models of A375 cells and HUVECs.Conclusion The NOP14 expression is negatively correlated with MVD in melanoma tissues,and NOP14 can inhibit angiogenesis in melanoma.
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Objective To evaluate the effect of overexpression of the autophagy marker gene Beclin I on biological behaviors of SK-MEL-2 human malignant melanoma cells.Methods Western blot analysis was performed to determine the protein expression of Beclin 1 in melanoma cell lines A375 and SK-MEL-2.SK-MEL-2 cells with low Beclin1 protein expression were selected as research objects,and divided into 3 groups:blank group receiving no treatment,negative control group transfected with pcDNA.3.1/myc-His (-) A,and experimental group transfected with pcDNA3.1-Beclin1 plasmid.After 2-week culture,cell counting kit-8 (CCK-8) assay was conducted to evaluate the effect of Beclin1 on cell proliferation at 24,48 and 72 hours,and Transwell assay and wound-healing assay were performed to assess the effect of Beclin 1 overexpression on the invasion and migration abilities of SK-MEL-2 cells.Repeated measures analysis of variance and completely randomized analysis of variance were used to analyze differences in indices among groups,and least significant difference (LSD)-t test was used for multiple comparisons.Results The protein expression of Beclin1 was significantly lower in the SK-MEL-2 cells (0.037 ± 0.010) than in the A375 cells (0.670 ± 0.150,F =46.62,P ＜ 0.05).The experimental group showed significantly increased protein expression of Beclin1 (0.32 ± 0.04) compared with the negative control group (0.06 ± 0.02,P ＜0.05) and blank group (0.07 ± 0.02,P ＜ 0.05).CCK-8 assay revealed a significant difference in the cell proliferation rate among different groups and different time points (F =1 077.36,4 903.04 respectively,both P＜ 0.05),and there was a significant interaction between the transfection treatment and time (F =205.20,P ＜ 0.05).Transwell assay showed that the number of SK-MEL-2 cells crossing the chamber per high-power field (× 200) after 24-hour treatment was significantly lower in the experimental group (18.67 ±1.19) than in the negative control group (87.89 ± 6.05,P＜ 0.05) and blank group (86.78 ± 5.93,P ＜0.05).In the wound-healing assay,the cell migration distance was significantly shorter in the experimental group than in the blank group and negative control group at 24 and 48 hours (all P ＜ 0.05).Conclusion Beclin 1 overexpression can markedly inhibit the proliferation,invasion and migration of SK-MEL-2 cells.
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Objective To evaluate the effect of luteolin on the growth,migration and vasculogenic mimicry formation of a melanoma cell line B16.Methods In vitro cultured B16 melanoma cells were divided into 4 groups:low-,middle-and high-dose luteolin groups treated with 2.5,5,10 μmol/L luteolin respectively,and control group treated with 0.1％ dimethyl sulfoxide (DMSO).Scratch assay,Transwell invasion assay and vascular channel formation assay were performed to assess the migration,invasion of and vascular channel formation by melanoma cells.A model of subcutaneous transplanted B 16 melanoma was established in 12 C57 mice,which were randomly and equally divided into 4 groups:control group gavaged with ultrapure water,low-,middle-and high-dose luteolin groups gavaged with 10,20,40 mg/kg luteolin respectively every day.The above treatment for the tumor-bearing mice lasted till day 28,and then these mice were sacrificed.Meanwhile,the lung and tumor tissues of the mice were excised,and the growth,metastasis and vasculogenic mimicry of transplanted melanoma were observed.Immunofluorescence and immunohistochemical studies were performed to evaluate the effects of luteolin on the expression of vascular endothelial cadherin (VE-cadherin),vascular endothelial growth factor receptor 1 (VEGFR1),VEGFR2,matrix metalloproteinase-2 (MMP-2) and MMP-9 in the transplanted melanoma.Means were compared among several groups by using one-way analysis of variation or rank sum test.Results In vitro study showed that the relative scratch width at 48 hours significantly differed among the control group,low-,middle-and high-dose luteolin groups (0.47 ± 0.04,0.64 ± 0.04,0.73 ± 0.03,0.84 ± 0.04 respectively;F =34.51,P ＜ 0.001),and the migration ability of B16 cells was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.05).At 24 hours,there were significant differences in the number of cells crossing the Transwell membrane among the control group,low-,middle-and high-dose luteolin groups (281.00 ± 8.79,169.00 ± 15.35,92.00 ± 14.79 and 57.00 ± 13.72 respectively;F =275.30,P ＜ 0.001),and the invasive ability was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (P ＜ 0.01).Meanwhile,the number of formed vascular channels also differed among the above 4 groups (20.00 ± 2.77,11.00 ± 1.28,7.00 ± 1.86 and 2.00 ± 1.32 respectively;F =48.61,P ＜ 0.001),and the number of vascular channels was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.01).In vivo study showed that the tumor size significantly differed among the control group,low-,middle-and high-dose luteolin groups (5.10 ± 1.72,4.02 ± 2.13,2.98 ± 0.92,1.49 ± 1.13 cm3 respectively;F =28.76,P ＜ 0.001),and was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (t =3.86,7.11 and 13.06 respectively,all P ＜ 0.01).CD31-PAS double staining showed that the number of vasculogenic mimicry was significantly higher in the control group than in the low-,middle-and high-dose luteolin groups (all P ＜ 0.01).In vivo and in vitro studies both showed that the expression of vasculogenic mimicry-related markers in the cells or mouse tumor tissues was significantly lower in the high-dose luteolin group than in the control group (P ＜ 0.05).Conclusion Luteolin can effectively inhibit the growth,metastasis and vasculogenic mimicry formation of melanoma.
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Objective To investigate the role of C/EBP homologous protein (CHOP)-dependent endoplasmic reticulum stress signaling pathway in triptolide-induced apoptosis of A375 melanoma cells,and to explore its mechanisms.Methods In vitro cultured human A375 melanoma cells were divided into several groups:experimental groups treated with triptolide at different concentrations of 12.5,25,50,100 and 200 nmol/L,and negative control group receiving no treatment.After 24-hour treatment,changes in the morphology of A375 cells were observed under a light microscope.After 24-,48-and 72-hour treatment,cell counting kit 8 (CCK8) assay was performed to evaluate the inhibitory effect of triptolide on cell proliferation.Flow cytometry was conducted to detect the apoptosis of A375 cells after annexin V-fluorescein isocyanate/propidium iodide double-staining,and transmission electron microscopy to observe the changes in the morphology of the endoplasmic reticulum.Western blot analysis was performed to determine the protein expression of glucose-regulated protein GRP78,protein kinase R-like endoplasmic reticulum kinase (PERK),phosphorylated PERK (p-PERK) and CHOP after 24-hour treatment,as well as to observe the changes in protein expression of GRP78 after treatment over time.Real-time fluorescencebased quantitative PCR (qPCR) was conducted to measure the mRNA expression of GRP78,PERK and CHOP.Results After the treatment with triptolide,A375 cells became long and thin,appeared fusiform with less cytoplasm,and varied in size.Their shape was irregular,and there were many protuberances on the cell surface.CCK8 assay showed that triptolide at different concentrations had inhibitory effects on the proliferation of A375 cells after 24-,48-and 72-hour treatment,and the inhibitory effects varied with the concentrations of triptolide and the duration of treatment (all P ＜ 0.05).The 50％ inhibitory concentration (IC50) of triptolide at 24,48 and 72 hours were 308,83 and 55 nmol/L respectively.The apoptosis rate of A375 cells was significantly higher in the 12.5-,25-,50-,100-and 200-nmol/L triptolide groups (10.3％ ± 0.1％,14.6％ ± 0.8％,17.4％ ± 0.7％,21.1％ ± 1.0％ and 29.5％ ± 1.1％,respectively) than in the negative control group (3.3％ ± 0.4％,all P ＜ 0.05).After 24-hour treatment with 200 nmol/L triptolide,damaged endoplasmic reticula were observed by using transmission electron microscopy.After 24-hour treatment with triptolide at different concentrations,the protein expression of GRP78,p-PERK,PERK and CHOP all gradually increased with the increase of triptolide concentrations (P ＜ 0.05).However,after 24-,48-and 72-hour treatment,the protein expression of GRP78 gradually decreased over time (P ＜ 0.05).qPCR showed that the mRNA expression of GRP78,PERK and CHOP gradually increased with the increase of triptolide concentrations after 24-hour treatment.Compared with the negative control group,all the experimental groups showed significantly higher mRNA expression of GRP78,PERK and CHOP (P ＜0.05) except the 12.5-nmol/L triptolide group with similar mRNA expression of PERK.Conclusion Triptolide can induce endoplasmic reticulum stress,and the apoptosis of A431 cells was induced by CHOP-dependent endo-plasmic reticulum stress along with the increase of triptolide concentrations and treatment duration.
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Objective To evaluate the effect of fenretinide (4-HPR)-loaded liposomes (4-HPR-L) on the proliferation,apoptosis and migration of A375 and B16F10 melanoma cells.Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L.In vitro cultured A375 cells,as well as B16F10 melanoma cells,were divided into the following groups:blank control group treated with fresh culture medium alone,4-HPR groups treated with 4-HPR at concentrations of 0.1,1,15,30,50 and 70 mg/L separately,and 4-HPR-L groups treated with 4-HPR-L at concentrations of 0.1,1,15,30,50 and 70 mg/L separately.Cell counting kit-8 (CCK8) assay was conducted to detect cell proliferation,Hoechst33258 staining and flow cytometry to detect cell apoptosis,wound healing assay to evaluate cell migration ability,and laser scanning confocal microscopy to observe endocytic uptake of the liposomes.Statistical analysis was done by one-way analysis of variance (ANOVA) for intergroup comparison,and a two-sample t-test for comparisons between 2 groups with SPSS 20.0 software.Results Both 4-HPR and 4-HPR-L showed inhibitory effects on the proliferation of A375 and B16F10 melanoma cells.After 48-hour treatment,the survival rates of A375 cells in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups were (94.3 ± 1.4)％,(91.7 ± 2.5)％,(84.4 ± 2.5％),(78.8 ± 2.1)％,(59.0 ± 1.1)％ and (42.8 ± 2.0)％ respectively,and those in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR-L groups were (86.0 ± 0.2)％,(76.5 ± 0.6)％,(60.9 ± 1.5)％,(49.0 ± 0.5)％,(32.9 ± 0.2)％ and (18.9 ± 0.5)％ respectively.After 48-hour treatment,the survival rates of B16F10 cells in 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups were (95.4 ± 1.9)％,(90.5 ± 2.6)％,(77.0 ± 0.8％),(64.4 ± 3.5)％,(59.1 ± 2.9)％ and (49.9 ± 1.9)％ respectively,and those in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR-L groups were (88.4 ± 2.0)％,(80.9 ± 3.4)％,(60.9 ± 2.2)％,(51.5 ± 2.9)％,(41.1 ± 1.2)％ and (33.5 ± 2.4)％ respectively.The survival rates of A375 and B16F10 cells were significantly higher in the 0.1-,1-,15-,30-,50-and 70-mg/L 4-HPR groups than in the 4-HPR-L groups at the same concentrations (A375 cells:t =8.019,8.298,11.455,19.978,33.672,16.314 respectively,all P ＜ 0.01;B16F10 cells:t =3.573,3.153,9.953,4.019,8.097,7.53 respectively,all P ＜ 0.05).Hoechst33258 staining showed that the morphology of the melanoma cells in the control group and 4-HPR groups did not change obviously,but the cells.in the 4-HPR-L groups became smaller,with the cytoplasm concentrated,nuclei dissociated into fragments,and apoptotic bodies formed.Flow cytometry showed that the apoptosis rates of A375 and B16F10 cells were significantly higher in the 4-HPR-L groups than in the 4-HPR groups (all P ＜ 0.01).Cell wound healing assay showed that the inhibitory effect of 4-HPR-L on the migration of cells was stronger than that of 4-HPR,and 4-HPR-L could markedly decrease the degree of wound healing.Laser scanning confocal microscopy showed that C6 liposomes could be rapidly and successfully internalized into both A375 and B16F10 cells.Conclusion The 4-HPR-L can be internalized into A375 and B16F10 cells better than 4-HPR,effectively inhibit the proliferation and migration of A375 and B16F10 cells,and induce the apoptosis of these cells.
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Objective To evaluate the effect ofT-cell immunoglobulin and mucin domain-3 (TIM-3) on TRP-2180-188 peptide-stimulated murine spleen lymphocytes co-cultured with B16F10 murine melanoma cells.Methods A recombinant plasmid pFUSE-TIM-3-mIgG2Aae1-Fc2 encoding TIM-3 was constructed.Then,the recombinant plasmid and an empty plasmid pFUSE-mIgG2Aae1-Fc2 were transfected into human 293T epithelial cells followed by 48-hour culture for the preparation of supernatants containing TIM-3 and Ig-tail respectively.C57BL/6 mice were immunized with the TRP-2180-188 peptide vaccine for 4 sessions.One week after the last vaccination,C57BL/6 mice were sacrificed,and spleen lymphocytes were collected and then cultured with the TRP-21180-188 peptide and interleukin-2 (IL-2) for 5 days,with lymphocytes untreated with the TRP-2180-188 peptide or IL-2 serving as the control group.Mitomycin-treated B16F10 murine melanoma cells and TRP-2180-188 peptide-stimulated lymphocytes were co-cultured with the presence of supernatants of 293T cells that had been cultured for 48 hours (blank control group),TIM-3-containing supernatants (TIM-3 group) and Ig-tail-containing supernatants (negative control group) separately.After 24 and 48 hours of co-culture,cell counting kit-8 (CCK-8) assay was performed to estimate the proliferative activity of lymphocytes,enzyme-linked immunosorbent assay (ELISA) to determine the supernatant levels of interferon (INF)-γ and tumor necrosis factor (TNF)-α,flow cytometry to determine the percentage of CD8 + T cells in the co-culture system.Results Enzyme digestion and sequence analysis showed that the TIM-3 gene was successfully inserted into the eukaryotic expression plasmid.After 48-hour culture,TIM-3 and Ig-tail expressions were detected in the supernatants of 293T cells transfected with the recombinant plasmid and empty plasmid respectively.As CCK-8 assay showed,the proliferative activity of lymphocytes was significantly lower in the TIM-3 group than in the blank control group and negative control group after 24-and 48-hour culture (78.06％ ± 6.37％ vs.100.00％ ± 10.42％ and 108.70％ ± 9.90％ at 24 hours,42.93％ ± 5.93％ vs.100.00％ ± 6.24％ and 168.00％ ± 2.98％at 48 hours,all P ＜ 0.05),so was the ratio of cellular proliferative activity at 48 hours to that at 24 hours (all P ＜ 0.05).Compared with the blank control group and negative control group,the TIM-3 group showed significantly decreased supernatant levels of IFN-γ and TNF-α after 24-hour (IFN-γ:192.96 γ 5.05 ng/L vs.216.44 ± 7.85 ng/L and 223.67 ±7.79 ng/L,both P＜ 0.05;TNF-α:58.43 ± 0.26 ng/L vs.26.43 ± 0.01 ng/L and 86.85 ± 1.12 ng/L,both P＜ 0.05) and 48-hour culture (IFN-γ:54.95 ± 0.57 ng/L vs.230.06 ± 4.23 ng/L and 167.24 ± 3.33 ng/L,both P ＜ 0.05;TNF-α:30.23 ±0.26 ng/L vs.26.84 ± 0.20 ng/L and 45.34 ± 0.22 ng/L,both P ＜ 0.05).In addition,the median percentage of CD8+ T cells was significantly increased in the TIM-3 group compared with the blank control group and negative control group after 24-and 48-hour culture (3.30％ vs.0.421％ and 2.22％ at 24 hours,4.06％ vs.0.577％ and 0.691％ at 48 hours,all P＜ 0.05).Conclusion TIM-3 in vitro can suppress the proliferative activity of and secretion of IFN-γand TNF-α by lymphocytes,but increase the percentage of CD8 + T cells in the co-culture system of TRP-2180-188 peptide-stimulated lymphocytes and B16F10 cells.
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Objective To evaluate regulatory effects of glutamate receptor antagonists on the proliferation and migration of WM451LU malignant melanoma cells, and to explore their related mechanisms. Methods WM451LU cells at exponential growth phase were classified into 3 groups to be treated with the glutamate receptor antagonist MK?801 at 100μmol/L(MK?801 group), the glutamate receptor antagonist CPCCOEt at 10μmol/L(CPCCOEt group), or culture medium(control group). After 24?hour treatment, methyl thiazolyl tetrazolium(MTT)assay was performed to determine cell proliferation rates, scratch assay to evaluate the migration activity of cells, and Western?blot analysis to measure expression levels of proliferating cell nuclear antigen (PCNA), protein kinase Cα(PKCα) both on cell membrane and in cytoplasm, and phosphorylated mitogen?activated protein kinase(p?MAPK). Results After 24?hour treatment, cell proliferation rates were significantly decreased in the MK?801 group and CPCCOEt group compared with the control group(63%± 3.1%and 60%± 2.4%vs. 100%± 1.1%, both P<0.05). The scratch assay showed that cell?free zones in the control group gradually narrowed over time, and the scratch wound tended to close. However, the cell?free zones in the MK?801 group and CPCCOEt group narrowed more slowly compared with the control group, and were still wide after 24?hour culture with no obvious closure of the scratch. The MK?801 group and CPCCOEt group both showed significantly decreased expressions of PCNA(77.0% ± 5.4% and 72.0% ± 4.2% respectively), PKCα on the cell membrane(0.12 ± 0.02 and 0.14 ± 0.02 respectively), and p?MAPK(0.48 ± 0.03 and 0.36 ± 0.04 respectively) compared with the control group(PCNA:100.0%± 1.3%;PKCα:0.38 ± 0.01;p?MAPK:1.00 ± 0.02;all P<0.05).Conclusion In vitro suppression of glutamate receptors can inhibit the proliferation and migration of WM451LU cells, likely through the mediation of the PKCα?MAPK signaling pathway.
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Objective To investigate the effects of an ar?turmerone derivative(ATD)on the proliferation and apoptosis of A375 human melanoma cells. Methods Both A375 cells and human skin fibroblasts (HSFs) were cultured with different concentrations(5, 10, 20, 40 and 80μmol/L)of ATD, vincristine and ar?turmerone, separately, for 48 hours in vitro. Subsequently, cell counting kit?8 (CCK?8) was used to evaluate cell proliferation, inverted microscopy to observe cell morphology after acridine orange/ethidium bromide (AO/EB) staining, and a colorimetric method to estimate caspase?3 activity. DNA fragmentation assay and flow cytometry were performed to assess cell apoptosis, and flow cytometry was conducted to analyze cell cycle. Results ATD, vincristine and Ar?turmerone all inhibited the proliferation of A375 cells in a dose?dependent manner(ATD:R2=0.99, F=340.96, P<0.05;vincristine:R2=0.99, F=349.19, P<0.05;ar?turmerone:R2=0.89, F=25.41, P<0.05). The fifty percent inhibitory concentra?tions(IC50s)of ATD, vincristine and ar?turmerone against A375 cells were 15.96 ± 0.02μmol/L, 77.00 ± 0.04μmol/L and 356.95 ± 0.01μmol/L respectively. When the drug concentrations were 5 and 10μmol/L, the proliferation of HSFs was inhibited by 8%± 0.06%and 25%± 0.02%respectively by ATD, by 49%± 0.09%and 34%± 0.07%respectively by ar?turmerone, and by 33%± 0.04%and 29%± 0.08%respectively by vincristine, and the proliferation of A375 cells was inhibited by 26%± 0.06%and 39%± 0.02%respectively by ATD, by 6%± 0.09%and 10%± 0.07%respectively by ar?turmerone, and by 8% ± 0.04% and 17% ± 0.08% respectively by vincristine, with the inhibitory effects of the three drugs being significantly different from that of dimethyl sulfoxide(all P<0.05). ATD showed stronger inhibitory effects on the proliferation of A375 cells, but weaker cytotoxic effects on HSFs compared with ar?turmerone and vincristine(all P<0.05). Meanwhile, ATD, vincristine and ar?turmerone all induced the apoptosis of A375 cells(P<0.05), and caspase?3 activity increased with the increase in drug concentrations(ATD:R2=0.98, F=162.30, P<0.05;vincristine:R2=0.96, F=94.39, P<0.05;ar?turmerone:R2=0.95, F=57.35, P<0.05). The effect of ATD on caspase?3 activity was strongest, followed by that of vincristine and ar?turmerone. As flow cytometry showed, all the three drugs induced cell apoptosis to different degrees, and ATD showed a relatively strong effect on cell apoptosis, especially late apoptosis, compared with the other two drugs. In the ATD group, the number of A375 cells in G1 phase gradually increased, while that in G2 phase and S phase significantly decreased with the increase in drug concentrations. Conclusions ATD exhibited proliferation?inhibiting and apoptosis?inducing effects on A375 cells, and the effects were stronger than those of vincristine and ar?turmerone. It is quite possible that ATD affects cell proliferation and differentiation by activating caspase?3 and arresting cell cycle in the G1 phase.
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Objective To evaluate effects of simultaneous inhibition of mammalian target of rapamycin complex 1(mTORC1)kinase and glycogen synthase kinase-3β(GSK-3β)on phosphorylation of 4E-binding protein-1(4EBP1), cap-dependent translation, as well as survival and apoptosis of melanoma cells. Methods Cultured A375 cells were classified into several groups to be treated with dimethyl sulfoxide (DMSO group), the mTORC1 kinase inhibitor everolimus at a concentration of 5 nmol/L (everolimus group), the GSK-3β kinase inhibitor AR-A014418 at a concentration of 10 μmol/L (AR-A014418 group), or 5 nmol/L everolimus and 10 μmol/L AR-A014418(combined treatment group). After additional culture, Western-blot analysis was performed to measure protein expressions of phosphorylated 4EBP1 (p4EBP1)and survivin in A375 cells, m7GTP pull down assay to estimate interaction between eukaryotic initiation factor-4E (eIF4E)and eIF4G, cell counting kit 8 (CCK8)assay to evaluate cell proliferation, and flow cytometry to detect cell apoptosis. Results Both everolimus and AR-A014418 had inhibitory effects on 4EBP1 phosphorylation and survivin expression. The expressions of p4EBP1-65 and survivin were both significantly decreased in the everolimus group (0.74 ± 0.05 and 0.71 ± 0.06 respectively), AR-A014418 group (0.62 ± 0.06 and 0.58 ± 0.07 respectively)and combined treatment group (0.14 ± 0.04 and 0.09 ± 0.05 respectively)compared with the DMSO group (1.00 ± 0.07 and 1.00 ± 0.06, respectively, all P < 0.001), with the most significant decrease observed in the combined treatment group. As m7GTP pull-down assay showed, the everolimus group, AR-A014418 group and combined treatment group all showed significantly lower relative expression levels of eIF4G(0.72 ± 0.04, 0.67 ± 0.05 and 0.12 ± 0.05 vs. 1.00 ± 0.06, all P < 0.001), but significantly higher relative expression levels of 4EBP1 (1.98 ± 0.16, 2.32 ± 0.17 and 7.58 ± 0.25 vs. 1.00 ± 0.08, all P < 0.001)than the DMSO group, and the combined treatment group showed the lowest eIF4G expression but highest 4EBP1 expression. After 24-hour culture, the proliferation of A375 cells was inhibited by 18.5% ± 1.3% in the everolimus group, 19.8% ± 1.8% in the AR-A014418 group, and 61.2% ± 2.1% in the combined treatment group compared with the DMSO group, with the strongest inhibition noted in the combined treatment group. The inhibitory effects of everolimus and AR-A014418 on cell proliferation increased over time, and showed the same trend at 48 hours. Flow cytometry showed that the apoptosis of A375 cells was accelerated by the 24-hour treatments with everolimus and AR-A014418 alone or in combination, with the apoptosis rate being 14.28% ± 2.18%, 14.57% ± 2.35% and 55.18% ± 6.27% in the everolimus group, AR-A014418 group and combined treatment group respectively, and the combined treatment showed the strongest accelerating effect. Conclusion The combined treatment with everolimus and AR-A014418 can evidently inhibit 4EBP1 phosphorylation and eIF4F complex formation in A375 cells, which then suppress cap-dependent translation and promote apoptosis of melanoma cells.
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Objective To explore molecular mechanisms underlying the in vitro counteracting effect of curcumin on malignant melanoma.Methods Cultured A375 and C8161 human melanoma cells were cultivated in vitro,and randomly divided into several test groups and a control group to be treated with different concentrations of curcumin and dimethyl sulfoxide respectively for different durations.Then,methyl thiazolyl tetrazolium (MTT) assay,Transwell assay,flow cytometry and Western blot were performed to evaluate the effect of curcumin on the proliferation,invasion and cell cycle of,as well as expressions of AKT/mTOR signaling pathway-related proteins in A375 and C8161 cells respectively.Statistical analysis was carried out by using t test.Results MTT assay showed that the treatment with curcumin of 5-35 mg/L for 24-96 hours significantly inhibited the proliferation of both A375 and C8161 cells compared with that with dimethyl sulfoxide (all P ＜ 0.001),and the inhibitory effect was in a dose-dependent manner within the range of 5-15 mg/L for A375 cells and within the range of 5-10 mg/L for C8161 cells,and in a time-dependent manner from 0 to 48 hours for both cells.After treatment for 24 hours,the 50％ inhibitory concentration (IC50) of curcumin against A375 cells and C 8161 cells was 10 mg/L and 5 mg/L respectively.Transwell assay demonstrated that the invasion of A375 and C8161 cells was significantly suppressed by 72-hour treatment with curcumin at 10 mg/L and 5 mg/L respectively (both P ＜ 0.001).Flow cytometry showed that the cell cycle of A375 and C8161 cells was arrested at G2/M phase after 24-hour treatment with curcumin at 10 mg/L and 5 mg/L respectively,with significant differences in the proportion of A375 cells and C8161 cells in G2/M phase between the test group and control group (A375 cells:35.00％ ± 3.54％ vs.120.80％ ± 7.46％,P＜ 0.001;C8161 cells:19.33％ ± 4.04％ vs.85.00％ ± 9.53％,P ＜ 0.001).Western blot revealed that the expressions of AKT/mTOR signaling pathway-related proteins were decreased in A375 and C8161 cells after 24-hour treatment with 10 mg/L and 5 mg/L curcumin respectively.Conclusion Curcumin can inhibit the proliferation and invasion of A375 and C8161 cells,likely by blocking cell cycle and inhibiting activation of the AKT/mTOR signaling pathway.
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Objective To investigate the effects of 2-methoxyestradiol (2-ME) on the proliferation and apoptosis of a mouse malignant melanoma cell line B16,and to explore their mechanism.Methods B16 cells were cultured in vitro,and divided into a negative control group receiving no treatment and several intervention groups treated with 2-ME at final concentrations of 5,10,20,40 mmol/L,respectively.After different durations of treatment,inverted phase-contrast microscopy was conducted to observe the morphologic change of B16 cells,sulforhodamine B (SRB) assay to evaluate proliferative activity and to draw growth curve of B16 cells according to the absorbance value at 490 nm,flow cytometry to detect cell cycle and apoptosis,and reverse transcription PCR and real-time PCR were performed to measure the expressions of the apoptosis-inducing gene gadd45b and proto-oncogene c-myc.Results As repeated measures analysis of variance showed,there were significant differences in the inhibitory effect on B16 cell proliferation among different concentrations (5,10,20,40 mmol/L) and different treatment durations (24,48,72 hours) of 2-ME (F =1170.94,1843.04,respectively,both P ＜ 0.01),and there was a significant interaction effect between these concentrations and treatment durations (F =272.79,P ＜ 0.01).After 48-hour treatment with 2-ME at 10,20 and 40 mmol/L,the apoptosis rate of B16 cells was increased to (4.13 ± 1.12)％,(11.25 ± 2.380)％ and (19.46 ± 2.9)％ respectively,compared to (0.23 ± 0.5)％ in the negative control group (all P＜ 0.01); the proportion of B16 cells in G0/G1 phase was increased to (59.5 ± 5.6)％,(63.4 ± 8.2)％ and (70.8 ± 4.4)％ respectively,compared to (44.1 ± 3.4)％ in the negative control group.There was a significant difference in the proportion of B16 cells in G0/G1 phase among the negative control group and intervention groups (F =13.56,P ＜ 0.05).Moreover,the mRNA expression of gadd45b was significantly enhanced after 24-hour treatment with 2-ME at concentrations of 20 and 40 mmol/L (both P＜ 0.01),while that of c-myc was significantly weakened after treatment with 2-ME at 10,20 and 40 mmol/L (all ＜ 0.05) compared with the negative control group.Conclusion 2-ME can inhibit the proliferation of B16 cells in vitro,upregulate the expression of gadd45b gene and downregulate the expression of C-myc gene.
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Objective To explore the effects of targeted silencing of the chemokine receptor 7 (CXCR7)gene on the invasion and migration of the melanoma cell line M14. Methods Western-blot analysis was performed to determine the protein expression of CXCR7 in melanoma cell lines M14 and A375, and CXCR7-overexpressing M14 cells were used in this study. Cultured M14 cells were divided into three groups: experimental group transfected with a small interfering RNA(siRNA)targeting CXCR7(CXCR7-siRNA), negative control group transfected with a negative control siRNA, blank control group receiving no treatment. Real-time quantitative PCR and Western-blot analysis were conducted to determine the mRNA and protein expressions of CXCR7 respectively in M14 cells, Transwell chambers were used to evaluate the invasive activity of M14 cells, and wound healing assay to estimate the migratory activity of M14 cells. Results The experimental group showed significantly decreased mRNA and protein expressions of CXCR7 compared with the negative control group and blank control group (CXCR7 mRNA: 0.412 ± 0.023 vs. 1.211 ± 0.117 and 1.000 ± 0.102, F = 30.068, P = 0.001; CXCR7 protein: 0.144 ± 0.005 vs. 1 and 1.016 ± 0.004, F =11 485.5, P = 0.000). The number of M14 cells crossing the polycarbonate membrane per high-power field (× 200)was significantly smaller in the experimental group than in the negative control group and blank control group (20.617 ± 1.503 vs. 42.000 ± 6.018 and 43.627 ± 2.152, F = 32.416, P = 0.001). Similarly, the number of migrating M14 cells in wound healing assay was significantly decreased in the experimental group compared with the negative control group and blank control group (15.00 ± 1.10 vs. 44.90 ± 2.20 and 45.30 ± 2.30, F = 2 411.945, P = 0.000). Conclusion Targeted silencing of the CXCR7 gene can significantly inhibit the invasion and migration of M14 cells in vitro, which may provide a potential target for the treatment of cutaneous melanoma.
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Objective To estimate the effect of a cis-imidazoline derivative,nutlin-3,on the biological behavior of A375 human melanoma cells,and to investigate its mechanism.Methods Cultured A375 cells were divided into several test groups treated with nutlin-3 at different concentrations (2.5,5,10 μmol/L) for 24,48 and 72 hours,and a control group treated with dimethyl sulfoxide (DMSO) only.Then,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity,Western blot to measure the expression of p53 protein,flow cytometry to estimate cell cycle phase distribution and apoptosis rate,and Transwell assay to evaluate migratory activity,of A375 cells.Statistical analysis was carried out by repeated-measures analysis of variance (ANOVA).Results After treatment with nutlin-3 of 2.5,5 and 10 μmol/L for 24,48 and 72 hours,significant differences were observed among different time points at each concentration and among different concentrations at the same time point in proliferation inhibition rate (F =67.43,135.58,respectively,both P ＜ 0.01),p53 protein expression level (F =1255.00,9196.00,respectively,both P ＜ 0.01),percentage of cells at G2 phase (F =831.38,267.99,respectively,both P ＜ 0.01),apoptosis rate (F =809.45,723.83,respectively,both P ＜ 0.01),migration inhibition rate (F =1100.00,1667.00,respectively,both P ＜ 0.01).The influence of nutlin-3 on cellular proliferative activity increased with the increase in its concentration,and that on percentage of cells at G2 phase,apoptosis rate and migratory activity increased with the increase in its concentration and treatment duration.There was a significant interaction between the treatment duration and concentration of nutlin-3 for p53 protein expression level in (F =826.79,P ＜ 0.01),percentage of cells at G2 phase in (F =21.602,P ＜ 0.01),apoptosis rate in (F =44.48,P ＜ 0.01),migratory activity of (F =313.09,P ＜ 0.01),and cellular proliferative activity of (F =26.95,P ＜ 0.01),A375 cells.Conclusion Nutlin-3 may inhibit the proliferation and migration of,but promote cell cycle arrest and apoptosis in,A375 cells,through accumulation of p53 protein.
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Objective To study the effect of paeonol on the proliferation and apoptosis of A375 human melanoma cells and its mechanism.Methods Cell counting kit-8 (CCK8) was used to evaluate the proliferative activity of A375 cells treated with paeonol of 0.5,1,2,4,8 mmol/L for 24,48 and 72 hours respectively.Subsequently,A375 cells were treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours followed by double staining with annexin V and propidium iodide for the detection of cell apoptosis,fluorometric assay for the estimation of caspase 3,caspase 8 and caspase 9 activity,and Western blot for the determination of the levels of p53,nuclear factor-κB proteins and some of their target proteins.The A375 cells receiving no treatment served as the blank control group.Statistical analysis was carried out by t test.Results Within the investigated concentration and time ranges,paeonol significantly inhibited the proliferative activity of A375 cells in a concentration-and timedependent manner.Compared with the blank control group,a significant increase was observed in the early apoptosis rate in A375 cells treated with paeonol of 1.25,2.5 and 5 mmol/L for 24 hours (13.74％-± 1.73％,25.95％ ± 0.57％ and 46.44％ ± 0.81％ vs.3.11％ ± 0.53％,P ＜ 0.05 or 0.01),as well as in the activity of caspase 3,8 and 9 in A375 cells treated with paeonol of 2.5 and 5 mmol/L for 24 hours (P ＜ 0.05 or 0.01).After 24-hour treatment,the protein levels of p53 and Bax were elevated,but those of nuclear factor-κB,Bcl-2 and Bcl-XL were decreased in A375 cells with the increase of paeonol concentration.Conclusions Paeonol can inhibit the proliferation but induce the apoptosis of A375 cells,and the apoptosis-inducing effect may be realized through intrinsic and extrinsic pathways by modulating nuclear factor-κB and p53 genes.
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Objective To study the effects of midkine (MK) gene-targeting small interfering RNA (siRNA)on the invasion of melanoma cells.Methods Three MK gene-targeting siRNAs (S1,S2 and S3)were designed,constructed,and transfected into human A375 melanoma cells.Real-time PCR was performed to measure the expression of MK gene and to screen the siRNA with best efficacy.Then,A375 cells were transfected with the optimal siRNA of various doses (3.125,6.25 and 12.5 nmol/L)followed by additional culture of various durations(24,48,72 hours).Some A375 cells remaining untreated served as the blank control group,and some transfected only with liposomes served as the vector control group.Reverse transcription (RT) -PCR and Western blot were conducted to detect the mRNA and protein expression of MK,respectively,MTT assay to observe the adhesion of A375 cells,and Boyden chamber was used to evaluate cell invasion.Results The expression of MK mRNA was downregulated by all the three siRNAs,especially by the siRNA S3,which was used in the following transfection experiment.Real-time quantitative PCR revealed that the MK mRNA expression was reduced by the siRNA in a dose- (r24hours=-0.906,r4Bhours=-0.922,r72hours=-0.939,all P<0.01)and time-dependent(r3.125nmol/L=-0.889,r625nmol/L=-0.935,r125nmol/L=-0.928,all P<0.01)manner.MTT assay showed that the percentage of adhesing cells was 73.66%±2.25%,49.36%±2.16%and 28.35%±1.68%in A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively.The number of cells migrating across the chamber filter was 23.9±1.6,12.1±1.5,5.6±1.2 among A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively,significantly lower than that in the blank control group(36.8±1.5).The percentage of adhesing cells and number of migrating cells decreased with the dose of siRNA(r=-0.936,-0.915,P<0.01,0.05,respectively).Conclusions MK gene might play an important role in the adhesion and invasion of melanonla cells.To down-regulate the expression of MK gene by siRNA may suppress the adhesion and invasion of melanoma cells.
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Objective To investigate the action mechanism of glutamate-mediated signaling pathway in malignant melanoma. Methods WM451LU melanoma cells in log phase were classified into 6 groups, negative control group treated with PBS (100 μl), MK801 group treated with the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (100 μmol/L), CPCCOEt group treated with non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonist CPCCOEt, MAP2 group transfected with adenovirus vector containing microtubule associated protein 2a (Ad-MAP2a), MK801 + MAP2 group treated with MK801 of 100 μmol/L and transfected with Ad-MAP2a, CPCCOEt + MAP2 group treated with CPCCOEt of 10 μmol/L and transfected with Ad-MAP2a. Western blot was performed to detect the expression of an ionotropic glutamate receptor, i.e., N-methyl-D-aspartate receptor type 2A (NMDAR2A) in WM451LU cells transfected with Ad-MAP2a. Scratch motility assay and cell invasion assay were conducted in vitro to detect the changes in migration and invasion ability of WM451LU cells after treated with Ad-MAP2a, MK-801, CPCCOEt alone or in combination. In vivo study was carried out to compare the inhibitory effect of the above treatments on melanoma. Results Western blot revealed a decrease in the expression of NMDAR2A in WM451LU cells after transfected with Ad-MAP2a. The scratch motility assay showed that the number of migrating cells per high power field was 117.04 ± 2.76 in MAP2 group,107.64 ± 6.50 in MK801 group,97.36 ± 4.79 in CPCCOEt group, 43.28 ± 3.02 in MK801 + MAP2 group,30.76 ± 3.97 in CPCCOEt + MAP2 group,significantly different from that in the negative control group (152.3 ± 5.75,all P ＜ 0.01 ). Cell invasion assay demonstrated that the average number of invading cells per high power field in the negative control was significantly higher than that in MAP2 group, MK801 group, CPCCOEt group, MK801+MAP2 group and CPCCOEt + MAP2 group (170.43 ±8.72 vs. 98.26 ± 3.84, 97.22 ± 5.54, 112.23 ± 7.21, 42.89 ± 5.06, 58.25 ± 6.68, P ＜ 0.05, 0.05, 0.05, 0.01and 0.01, respectively).A significant decrease was observed in the average volume of experimental melanoma in mice of MAP2 group, MK801 group, MK801 + MAP2 group, CPCCOEt group and CPCCOEt + MAP2 group compared with the negative control group (224.02 ± 46.19 mm3, 160.33 ± 33.91 mm3, 91.49 ± 21.48 mm3,202.30 ± 52.37 mm3, 111.13 ± 69.81 mm3 vs. 342.70 ± 60.92 mm3, all P ＜ 0.01 ). Conclusions To block the glutamate signaling pathway in vitro can inhibit the invasion and migration of melanoma cells, and to block the pathway in vivo can inhibit the growth of malignant melanoma and alter the morphology of melanoma cells.
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Objective To investigate the modulation of ET-3 on the nuclear factor (NF)-κB/Bfl-1 antiapoptotic pathway in a malignant melanoma cell line A375. Methods Flow cytometry was performed to detect the apoptosis in cultured A375 cells after treatment with ET-3 of 100 nmol/L for 24 hours. ET-3 of various concentrations (0, 1, 10, 100 nmol/L) was used to treat some A375 cells with or without the pretreatment with the ETRB antagonist BQ788; after another 24-hour culture, RT-PCR and Western blot were conducted to examine the mRNA expression of Bfl-1 and protein expressions of Bfl-1 and ETRB, respectively. Results The 24-hour treatment with ET-3 of 100 nmol/L significantly reduced the apoptosis rate of A375 cells (F = 10.68, P ＜0.05). The mRNA and protein expressions of Bfl-1 were up-regulated by ET-3 in a concentration dependent manner (both P ＜ 0.01 ), while BQ788 significantly blocked the ET-3-induced up-regulation (F = 420.38,229.49, both P ＜ 0.01 ). The protein expression of pNF-κB in A375 cells was also enhanced by ET-3 of different concentrations (all P ＜ 0.05), but the enhancement was suppressed by BQ788, and there was a significant difference in the protein expression of pNF-κB between cells treated with ET-3 of 100 nmol/L and those treated with the combination of ET-3 of 100 nmol/L and BQ788 (F = 255.46, P ＜ 0.01 ). Conclusion ET-3/ETRB inhibits the apoptosis in A375 cells likely by activating the NF-κB/Bfl-1 anti-apoptotic pathway.
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Objective To construct an artificial skin model of melanoma by mixed culture of human keratinocytes and MV3 melanoma cells on de-epidermized dermis (DED) in order to study the effect of keratinocytes on melanoma invasion. Methods Epidermal cell suspension was obtained by a two-step digestion method from the circumcised foreskin of a child, keratinocyte serum-free medium was applied to the culture and passage of keratinocytes. MV3 melanoma cells were cultured and passaged in RPMI 1640 medium. Log-phase keratinocytes and MV3 cells were mixed with a ratio of 3:1 and seeded onto the surface of DED followed by a liquid culture and air-liquid culture for a total of 2 weeks. Thereafter, the artificial tissue model was assessed by HE staining and immunohistochemical staining for S-100 protein, HM64S and keratin. Results HE staining showed that MV3 cells formed band-like tumor masses or foci on the surface of DED, with keratinocytes intermingling among the tumor cells, but no typical epidermis-like structure was observed. Some tumor cells infiltrated into the surface of DED and showed a cluster distribution; some tumor cells invaded the lumen of the DED, and attached to the luminal wall in a ring shape; some tumor cells penetrated through the wall into the surrounding dermal tissue. On the bottom and lateral side of DED, tumor cells were infiltrating dispersedly.The tumor loci stained positive for S-100 protein and keratin, and weakly positive for HMB45. Conclusion Keratinocytes enhance the invasion of MV3 melanoma cells into the skin tissue model of melanoma.