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Objective:To investigate the effect of long non-coding RNA 068 (lncRNA 068) on the migration of a melanoma cell line A375, and to explore its mechanism of action.Methods:From December 2015 to November 2020, 21 patients with pathologically confirmed cutaneous melanoma were collected from Department of Dermatology, Affiliated Hospital of Nantong University, and quantitative PCR (qPCR) was performed to determine the expression of lncRNA 068 in melanoma and paracancerous tissues. LncRNA 068 was overexpressed or knocked down via lentiviral transfection in A375 human melanoma cells in the following experiments. Specifically, A375 cells were divided into lentiviral vector (LV) -green fluorescent protein (GFP) group and LV-lncRNA 068 group to be transfected with a GFP-expressing LV and a LV containing lncRNA 068 respectively in the overexpression experiment, and were divided into LV-LacZ short hairpin RNA (shRNA) group and LV-lncRNA 068 shRNA group to be transfected with a LV containing the reporter gene LacZ-specific shRNA and a LV containing the lncRNA 068-targeting shRNA respectively in the low-expression experiment, with the LV-GFP group and LV-LacZ shRNA group serving as the control groups. Transwell and scratch assays were performed to evaluate cell migration, EdU cell proliferation assay and cell counting kit-8 (CCK8) assay to determine the proportion of proliferative cells and cell viability respectively, and immunofluorescence staining was conducted to evaluate epithelial-mesenchymal transformation in the above groups. Lentivirus-transfected A375 cells from the above groups were inoculated into the axillae of BALB/c nude mice, and tumor volume was measured and calculated every 3 days. After 30 days, all mice were sacrificed, and tumor tissues were resected to measure the tumor volume and weight. Cultured B16F10 cells were subcutaneously inoculated into the back and foot of BALB/c nude mice to construct mouse models of subcutaneously transplanted B16F10 melanoma. After 2 weeks, the mice were sacrificed, and qPCR and Western blot analysis were performed to determine the mRNA expression of inflammatory factors in transplanted B16F10 melanoma and paracancerous tissues, and expression of IκB kinase (IKK) /P65 signaling pathway-related proteins, respectively. Comparisons between 2 groups were done by t test, and comparisons of tumor volume and weight at different time points among groups were done by repeated measures analysis of variance. Results:qPCR showed that the relative expression of lncRNA 068 was significantly lower in human melanoma tissues and transplanted B16F10 murine melanoma tissues (0.414 ± 0.109, 0.717 ± 0.041, respectively) than in the corresponding paracancerous tissues (1.050 ± 0.103, 1.011 ± 0.023, t = 19.48, 10.83, respectively, both P < 0.001). Transwell and scratch assays both showed that the cellular migratory ability was significantly lower in the LV-lncRNA 068 group than in the LV-GFP group (both P < 0.01), and significantly higher in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group (both P < 0.05). Immunofluorescence assay showed significantly increased fluorescence intensity of E-cadherin and decreased fluorescence intensity of N-cadherin in the LV-lncRNA 068 group compared with the LV-GFP group (both P < 0.001), but significantly decreased fluorescence intensity of E-cadherin and increased fluorescence intensity of N-cadherin in the LV-lncRNA 068 shRNA group compared with the LV-LacZ shRNA group (both P < 0.05). In vivo tumor formation experiment in nude mice showed that there were no significant differences in the volume or weight of melanoma between the LV-lncRNA 068 group and LV-GFP group (both P > 0.05), as well as between the LV-lncRNA 068 shRNA group and LV-LacZ shRNA group (both P > 0.05). As qPCR and Western blot analysis showed, the mRNA and protein expression of interleukin-10 (IL-10) and claudin-1 in A375 cells were significantly higher in the LV-lncRNA 068 group than in the LV-GFP group (both P < 0.05), but significantly lower in the LV-lncRNA 068 shRNA group than in the LV-LacZ shRNA group (both P < 0.05). Compared with the paracancerous tissues, B16F10 melanoma tissues showed significantly decreased mRNA expression of IL-10 ( P < 0.01), but significantly increased mRNA expression of IL-6 and tumor necrosis factor-α, as well as protein expression of phosphorylated P65 and phosphorylated IKK ( P < 0.01) . Conclusion:Overexpression of lncRNA 068 can inhibit the migration of A375 melanoma cells, and may affect the development of inflammation and inhibit the epithelial-mesenchymal transformation by inhibiting the IKK/P65 signaling pathway.
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Objective:To investigate the effect of the methyltransferase inhibitor azacitidine (5-azaC) on the expression of homeobox A9 (HOXA9) gene in, as well as proliferation, invasion and migration of A375 cells.Methods:In vitro cultured A375 cells were treated with 5-azaC at various concentrations of 1, 5, 10 and 20 μmol/L, while routinely cultured A375 cells receiving no drug intervention served as control group. Methylation-specific PCR was performed to analyze methylation status of the HOXA9 gene promoter region after the treatment with different concentrations of 5-azaC, in order to screen the optimal concentration of 5-azaC for following experiments. Cell counting kit-8 (CCK8) assay was conducted to evaluate the proliferation of A375 cells, Transwell and wound healing assays were performed to estimate the invasion and migration of A375 cells, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of HOXA9 in A375 cells after 5-azaC treatment. Two-independent-sample t test was used for comparisons between two groups. Results:Methylation was observed in the HOXA9 gene promoter region in A375 cells in the control group. After 5-azaC treatment, methylated and unmethylated states coexisted in the HOXA9 gene promoter region in A375 cells, and the higher the concentration of 5-azaC, the higher the degree of demethylation of the HOXA9 gene. Therefore, 20 μmol/L 5-azaC was selected to treat A375 cells for 72 hours, which served as 5-azaC treatment group in subsequent experiments. Compared with the control group, the 5-azaC treatment group showed significantly decreased cellular proliferative ability (72.46% ± 2.19% vs. 100%, t = 28.09, P < 0.001) , significantly decreased number of invasive cells (242.70 ± 29.19 vs. 466.00 ± 22.65, t = 10.47, P < 0.001) , significantly decreased migratory ability (27.56% ± 2.74% vs. 35.69% ± 2.50%, t = 3.79, P = 0.019) , significantly increased HOXA9 mRNA expression (1.73 ± 0.28 vs. 1.01 ± 0.15, t = 3.93, P = 0.017) , and significantly increased HOXA9 protein expression (0.62 ± 0.03 vs. 0.50 ± 0.01, t = 3.82, P = 0.019) . Conclusion:5-azaC can inhibit the proliferative, invasive and migratory ability of A375 melanoma cells, and one of the possible mechanisms underlying this process may be that 5-azaC reverses the methylation in the HOXA9 gene promoter region, activates HOXA9 gene expression, and participates in the regulation of biological behaviors of melanoma cells.
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Objective To evaluate the inhibitory effect of fenretinide-loaded liposomes(4-HPR-L) on subcutaneous transplanted malignant melanomas in nude mice.Methods A film-ultrasonic dispersion method was used to prepare 4-HPR-L.BALB/c nude mice were subcutaneously inoculated with A375 melanoma cells in the right axillary fossae to establish malignant melanoma-bearing nude mouse models.Ten nude mouse models were randomly and equally divided into 2 groups to be injected with near-infrared fluorescent cell membrane label (DiR) solution or DiR liposomes (DiR-L)at the same concentration in the caudal vein,and a live imaging system was used to observe the distribution of DiR or DiR-L in nude mice at 6,12,24 hours after the injection.Another 30 nude mice were randomly and equally divided into 3 groups to be injected with 5％ (mass fraction) glucose solution at a single-dose of 0.2 ml (control group),25 mg/kg 4-HPR solution (4-HPR group)and 25 mg/kg 4-HPR-L solution (4-HPR-L group) respectively on days 8,10,12,14,16,18,20 and 22 after the inoculation with A375 cells.The mouse body weight and tumor volume were dynamically monitored in the above groups after the injection,and the survival situation was observed.The nude mice were sacrificed on day 2 after the final injection,and the heart,liver,spleen,lung,kidney and tumor tissues were resected.These tissues were subjected to hematoxylin-eosin staining and immunohistochemical staining to observe the metastasis of melanoma in mice,and terminal deoxyribonucleotidyl transferase-mediated nick end labelling was performed to detect the apoptosis in tumor cells.One-way analysis of variance and independent-sample t test were used to analyze measurement data.Results The live imaging system showed that DiR-L could be retained in melanoma for a long time,and strong fluorescence of DiR-L could be still observed in the tumors at 24 hours after injections.Quantitative fluorescence analysis revealed that the fluorescence intensity of DiR-L (22.85 ± 1.66) was significantly higher than that of DiR in tumor tissues (8.45 ± 0.97,t =12.957,P ＜ 0.01).Compared with the control group and 4-HPR group,the resected tumor weight on day 2 after the final injection was significantly decreased in the 4-HPR-L group (F =27.055,t =4.768,6.640,respectively,both P ＜ 0.05).Hematoxylineosin staining showed that liver metastasis occurred in 2 nude mice in the 4-HPR-L group,but in all the nude mice in the control group and 4-HPR group.All the nude mice in the 4-HPR-L group died within 76 days after inoculation,and the mice in the control group and 4-HPR group all died within 56 and 59 days respectively after inoculation.There were significant differences in the apoptotic index among the control group (12.14‰ ± 1.33‰),4-HPR group (67.17‰± 15.18‰) and 4-HPR-L group (152.73‰ ± 11.27‰;F =167.588,P ＜ 0.05),and the apoptotic index was significantly higher in the 4-HPR-L group than in the control group and 4-HPR group (t =18.162,11.075 respectively,both P ＜ 0.05).Conclusion 4-HPR-L can effectively inhibit the growth of subcutaneous melanoma in nude mice and metastasis of melanoma cells,and prolong the survival duration of nude mice.
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Objective: To prepare Juglone-loaded poly lactic-co-glycolic acid nanoparticles (Jug-PLGA-NPs), and investigate their physicochemical properties, release characteristics in vitro and anti-tumor activities on A375 melanoma cells in vitro. Method: Jug-PLGA-NPs were prepared by emulsification-solvent evaporation method. Then the particle size, encapsulation efficiency, drug loading rate and in vitro release characteristics were investigated. Fluorescence microscopy was used to observe the uptake of PLGA-NPs in vitro. The distribution of PLGA-NPs in BALB/c nude mice after tail vein injection was observed by the small living animal imaging system. Their inhibition effect on proliferation of A375 cells was detected by thiazolyl blue tetrazolium bromide (MTT) assay. Apoptosis rate and cell cycle detection were performed by flow cytometry. Western blot was used to determine the protein kinase B (Akt), phosphorylated Akt (p-Akt) and cyclinD1. Result: The average particle size of the prepared Jug-PLGA-NPs was (149.6±21.5) nm, entrapment rate of (68.39±2.51)%, and drug-loading rate of (5.07±0.98)%, showing good sustained-release characteristics. PLGA-NPs showed good penetration and targeting properties in cellular uptake in vitro and in vivo imaging. Different concentrations of Jug-PLGA-NPs could significantly inhibit the proliferation and promote apoptosis of A375 cells in a time and concentration dependent manner (P1 expression (P0/G1 phase (PConclusion: The Jug-PLGA-NPs are easy to prepare and have good sustained-release characteristics, tumor targeting and anti-tumor ability, providing a new pharmaceutical dosage form for the future clinical application of Jug.
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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.
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Objective To evaluate the effect of berberine on the expression of cell cycle-related miRNA and its target gene in human melanoma A375 cells.Methods In vitro cultured A375 cells were classified into several groups to be treated with berberine at different concentrations of 0 (blank control group),20,40,60,80,and 100 μmol/L,respectively,for 48 hours.qRT-PCR was performed to determine the mRNA expression of miRNA-582-5p and its target gene cyclin-dependent kinase 1 (CDK1),miRNA-188-5p and its target gene CDK2,Cyclin D1 and Cyclin A.Western blot analysis was conducted to measure the protein expression of cell cycle-related proteins CDK1,CDK2,Cyclin D1 and Cyclin A.Results qRT-PCR showed that compared with the blank control group,the 20 and 40 μmol/L berberine groups had a similar expression of miRNA-582-5p (both P ＞ 0.05),but the 60 and 80 μmol/L berberine groups had a significantly up-regulated expression of miRNA-582-5p (both P ＜ 0.05).Compared with the blank control group,all the 5 berberine groups had a significantly increased expression of miRNA-188-5p (F =22.600,P =0.002).However,the mRNA expression of CDK2,CDK1,Cyclin A and Cyclin D1 gradually decreased along with the increase of berberine concentrations (F =51.976,248.510,626.671 and 312.740,respectively,all P ＜ 0.001).Western blot analysis revealed that berberine decreased the protein expression of CDK1,CDK2,Cyclin D1 and Cyclin A (F =138.124,110.966,278.772 and 140.167,all P ＜ 0.001).Conclusion Berberine can decrease the expression of cell cycle-related proteins CDK1,CDK2,Cyclin D1 and Cyclin A,likely by decreasing their mRNA expression and increasing the expression of miRNA-582-5p and miRNA-188-5p,and then block the cell cycle progression of A375 cells and inhibit the growth of tumor.
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Objective To construct a recombinant lentiviral vector carrying the casitas B-lineage lymphoma (Cbl)-b short hairpin RNA (shRNA),and to evaluate its effect on the biological behavior of A375 melanoma cells in vitro.Methods Three specific shRNAs targeting Cbl-b gene and a negative control shRNA were designed and synthesized,and recombinant lentiviral vectors were constructed.A375 cells were divided into 5 groups to be transfected with 3 kinds of lentiviral vector expressing Cbl-b genespecific shRNAs (CBLB-shRNA-1 group,CBLB-shRNA-2 group and CBLB-shRNA-3 group),a lentiviral vector containing negative control shRNA (negative control group),and an empty vector (blank control group).Real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine the silencing efficiency at 72 hours after transfection.Cell counting kit-8 (CCK-8)assay was conducted to evaluate cellular proliferative activity at 24,48,72 and 96 hours after transfection,flow cytometry to detect cell apoptosis and cell cycle at 72 hours after transfection,and Transwell invasion assay to assess cellular invasive activity at 72 hours after transfection.Results Three recombinant lentiviral vectors containing Cbl-b shRNA were constructed successfully.As Western blot analysis revealed,the CBLB-shRNA-3 showed the highest silencing efficiency.CCK-8 assay indicated that the proliferative activity of A375 cells was significantly lower in the CBLB-shRNA-3 group than in the negative control group and blank control group at 72 and 96 hours after transfection(all P ＜ 0.01).Flow cytometry showed that the apoptosis rate of A375 cells was significantly higher in the CBLB-shRNA-3 group (22.73％ ± 6.58％) than in the negative control group (6.08％ ± 1.35％,P ＜ 0.01) and blank control group (6.34％ ± 1.07％,P ＜ 0.01).The CBLB-shRNA-3 group showed a significantly higher proportion of A375 cells at G1 phase,but a significantly lower proportion of A375 cells at S phase compared with the negative control group and blank control group(all P ＜ 0.01).Transwell assay showed that there were significant differences in the number of A375 cells crossing the artificial basement membrane (matrigel) at 72 hours after transfection among the negative control group,blank control group and CBLB-shRNA-3 group (76.60 ± 1.82,73.20 ± 3.83,19.60 ± 1.14,respectively;F =794.50,P ＜ 0.01).Conclusions A recombinant CBLB-shRNA-3-expressing lentiviral vector which can efficiently silence Cbl-b gene has been successfully constructed.It can inhibit the proliferation,cell cycle progression and invasive activity of A375 cells,but promote the apoptosis of A375 cells.
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OBJECTIVE: To investigate the effect of ultraviolet B( UVB) on autophagy and apoptosis in human epidermal melanoma A375 cells. METHODS: i) A375 cells at logarithmic growth phase were exposed to UVB at doses of 10. 0 and15. 0 m J/cm~2. Then cells were collected at time point of 3,6,9 and 12 hours after irradiation. The effect of UVB on cell autophagy was observed by monodansylcadaverine staining and the effect of UVB on cell apoptosis was observed by acridine orange/ethidium bromide staining. ii) A375 cells of 10. 0 m J/cm~2 group and 15. 0 m J/cm~2 group were exposed to corresponding dose of UVB irradiation. Then cells were collected at time point of 18,24,36 and 48 hours after irradiation,and cell survival rate was examined using CCK-8 assay. iii) A375 cells were irradiated with UVB at doses of 10. 0 and15. 0 m J/cm~2 and then cells were collected at time point of 3,6,9 and 12 hours after irradiation. After that,A375 cells were irradiated at doses of 2. 5,5. 0,7. 5,10. 0 and 15. 0 m J/cm~2 of UVB,then cells were collected at time point of 9 hours after irradiation. The expressions of B-lymphoblastoma-2( Bcl-2),Bcl-2 related X protein( Bax),Bcl-2 interacting protein( Beclin-1) and microtubule-associated protein 1 light chain 3( LC3) Ⅱ were detected by Western blotting. A375 cells with no UVB irradiation were set as the control( pseudo-irradiation) in each experiment. RESULTS: i) Both autophagy and apoptosis of A375 cells induced by UVB irradiation at doses of 10. 0 and 15. 0 m J/cm~2 increased with time after irradiation. The effect on autophagy decreased at 12 hours time point with 15. 0 m J/cm~2 UVB irradiation. ii) The cell viability increased with time after irradiation in the 10. 0 and 15. 0 m J/cm~2 groups( P < 0. 05). From 18-48 hours after irradiation,the cell viability of the 10. 0 and 15. 0 m J/cm~2 groups was lower than that of the control group( P < 0. 05).From 24-48 hours after irradiation,the cell viability of the 15. 0 m J/cm~2 group was lower than that of the 10. 0 m J/cm~2 group( P < 0. 05). iii) The relative expression of Beclin-1 and LC3 Ⅱ protein at the 10. 0 m J/cm~2 group increased with time after 0-12 hours irradiation( P < 0. 05). The above changes of the 15. 0 m J/cm~2 group were observed within 0 to 9 hours,and the above two autophagy-related proteins were significantly decreased at the 12 hours time point( P < 0. 05).The relative expression of Bcl-2 protein at the 10. 0 and 15. 0 m J/cm~2 groups decreased with increasing time from 3 to 12 hours after irradiation( P < 0. 05). The relative expression of Bax protein increased with time from 0 to 12 hours after irradiation( P < 0. 05). The relative expression of Beclin-1 and LC3 protein in cells at 0. 0-10. 0 m J/cm~2,and the relative expression of Bax protein in cells at 0. 0-15. 0 m J/cm~2 increased with increase of irradiation dose( P < 0. 05). The relative expression of Bcl-2 protein decreased with increase of irradiation dose at 5. 0-15. 0 m J/cm~2( P < 0. 05). CONCLUSION: Autophagy and apoptosis of A375 cells can be induced by UVB irradiation at doses of 10. 0 and 15. 0 m J/cm~2. Autophagy induced by UVB irradiation at 10. 0 m J/cm~2 partially resisted the induction of apoptosis by UVB and enhanced cell viability. 15. 0 m J/cm~2 UVB-induced autophagy was insufficient to exert the above-mentioned effects,and the induction of apoptosis was the dominant effect.
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Objective To investigate the expression of DKK3 in human cutaneous malignant melanoma cells and tissues,and to evaluate the effect of transfection with DKK3 gene on migration and invasion of a malignant melanoma cell line A375.Methods Western blot analysis was performed to measure the relative expression of DKK3 in human cutaneous melanoma cell lines HM,A375,WM451,SK-MEL-1,Hs-695T,MDA-MB-435s and WM35,as well as pigmented nevus tissues.Real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of DKK3 in 58 melanoma tissues (including primary melanoma and metastatic melanoma) and 30 pigmented nevus tissues from Chongqing Traditional Chinese Medicine Hospital between August 2014 and June 2017.The pcDNA3.1 (+)-Flag-Vector (control group) and pcDNA3.1 (+)-Flag-DKK3 (transfection group) were transfected into A375 melanoma cells separately.RT-PCR and Western blot analysis were performed to verify the overexpression of DKK3,and to evaluate the effect of DKK3 overexpression on the expression of molecules related to the migration and invasion of melanoma cells.Cell scratch assay,Transwell migration and invasion assay were conducted to assess the effect of DKK3 on the migration and invasion of A375 cells.Statistical analysis was done by a two-sample t-test for comparisons between two groups,one-way analysis of variance (ANOVA) for intergroup comparison,and least significant difference (LSD)-t test for multiple comparisons with the SPSS 13.0 software.Results DKK3 protein was absent or lowly expressed in the human melanoma cell lines,but highly expressed in the pigmented nevus tissues.There were significant differences in the mRNA expression of DKK3 among the primary melanoma tissues (2-ΔΔCt:[0.325 ± 0.150] × 10-3),metastatic melanoma tissues ([0.142 ± 0.210] × 103) and pigmented nevus tissues ([0.634 ±:0.120] × 10-3,F =46.57,P ＜ 0.05).In addition,the mRNA expression of DKK3 was significantly lower in the metastatic melanoma tissues than in the primary melanoma tissues and pigmented nevus tissues (LSD-t =2.48,3.12,both P ＜ 0.05).After transfection with DKK3,cell scratch assay showed that the migration rate was significantly lower in the transfection group (22.11％ ± 5.11％) than in the control group (54.36％ ± 23.22％,t =2.36,P ＜ 0.001).Transwell migration and invasion assay revealed that the number of A375 cells crossing the Transwell chamber was significantly lower in the transfection group (265 ± 33,76 ± 18 respectively) than in the control group (429 ± 41,135 ± 21 respectively;t =1.24,1.35 respectively,both P ＜ 0.001).After overexpression of DKK3 in the A375 cells in the transfection group,the mRNA and protein expression of E-cadherin were up-regulated,while the mRNA and protein expression of N-cadherin,vimentin,matrix metalloproteinase 2 (MMP2),MMP7 and MMP11 were down-regulated compared with the control group.Conclusions The expression of DKK3 is down-regulated in the melanoma cell lines and tissues,and the migration and invasion of A375 cells are markedly inhibited by overexpression of DKK3.DKK3 may be a target for inhibiting the metastasis of cutaneous malignant melanoma.
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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.
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Objective To investigate in vitro effects of tanshinoneⅡA on the autophagy of A375 melanoma cells and related signaling pathway. Methods Some cultured A375 cells were divided into 5 groups to be treated with tanshinoneⅡA at concentrations of 0.5, 1, 2 and 4 mg/L, and DMEM containing 0.1% dimethyl sulfoxide (DMSO), respectively, for 24, 48, 72 hours. Methyl thiazol tetrazolium (MTT) assay was performed to estimate the proliferative activity of A375 cells. Some cultured A375 cells were divided into 4 groups to be treated with 1, 2 and 4 mg/L tanshinone ⅡA(1?, 2?and 4?mg/L tanshinone group), and DMEM containing 0.1% DMSO (control group), respectively, for 48 hours. Then, flow cytometry was conducted to count autophagosome?positive cells, and Western blot analysis to determine protein expression of autophagy?associated proteins Beclin?1, microtubule?associated protein 1 light chain 3 (LC3)?Ⅱ, phosphatidylinositol 3?kinase(PI3K), protein kinase B(Akt), mammalian target of rapamycin (mTOR)and p70 ribosomal protein S6 kinase 1(p70S6K1). Results MTT assay showed that 24?, 48?, 72?hour treatments with tanshinone ⅡA at concentrations of 0.5, 1, 2 and 4 mg/L all could inhibit the proliferative activity of A375 cells, and the inhibitory effects increased in a dose? and time?dependent manner(F = 2 564.12, 1 235.25, both P < 0.05). The percentage of autophagosome?positive cells and protein expression of Beclin?1 and LC3?Ⅱincreased gradually and significantly in the 1?, 2?and 4?mg/L tanshinone groups(autophagosome?positive cells: 6.91% ± 0.35%, 13.11% ± 0.73%, 25.51% ± 0.83%, respectively; Beclin?1: 0.33 ± 0.01, 0.53 ± 0.04, 0.63 ± 0.02, respectively; LC3?Ⅱ: 0.41 ± 0.01, 0.52 ± 0.02, 0.64 ± 0.02, respectively), after 48?hour treatment, which were significantly different between the tanshinone groups(all P<0.05), and higher in the tanshinone groups than in the control group(0.41%±0.02%;0.09 ± 0.02;0.21 ± 0.01, all P<0.05). However, the protein expression of PI3K, phosphorylated Akt(p?Akt), p?mTOR and p?p70S6K1 in the PI3K?Akt?mTOR?p70S6K1 signaling pathway decreased gradually and significantly with the increase in tanshinone concentrations after 48?hour treatment, and were significantly lower in all the tanshinone groups than in the control group (all P < 0.05). Conclusion Tanshinone ⅡA can promote the auophagy of A375 cells, likely by blocking the PI3K?Akt?mtTOR?p70S6K1 signaling pathway.
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Objective To evaluate effects of tanshinone ⅡAon the invasion and migration of melanoma A375 cells,as well as on the mRNA and protein expression of CXC chemokine receptor type 7 (CXCR7).Methods In vitro cultured A375 cells were divided into 4 groups to be treated with tanshinone ⅡA at different concentrations of 1,2 and 4 mg/L,and 0.1％ dimethyl sulfoxide (DMSO) (control group) for 48 hours,respectively.Wound scratch assay and Transwell invasion assay were conducted to estimate the migratory and invasive abilities of A375 cells,respectively,and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis to determine the mRNA and protein expression of CXCR7 in A375 cells,respectively.Results After 48-hour treatment,the 1-,2-and 4-mg/L tanshinone ⅡA groups showed significantly decreased number of A375 cells crossing the polycarbonate membrane per high-power field (× 200) (71.00 ± 4.00,51.00-± 2.00 and 37.00 ± 3.61,respectively) in Transwell invasion assay,as well as decreased number of A375 cells migrating to the scratch zone (301 ± 3.00,253.00 ± 3.61 and 126.00 ± 7.00,respectively) in wound scratch assay,compared with the control group (105.33 ± 6.51,332.00 ± 6.24,respectively,all P ＜ 0.05).Additionally,qRT-PCR and Western blot analysis revealed that the mRNA and protein expression of CXCR7 was significantly lower in the 1-,2-and 4-mg/L tanshinone ⅡA groups than in the control groups (CXCR7 mRNA:0.63-± 0.04,0.44 ± 0.02 and 0.31 ± 0.01 vs.1.00 ± 0.02;CXCR7 protein:0.573 ± 0.015,0.416 ± 0.011 and 0.260-± 0.055 vs.0.9000 ± 0.010;all P ＜ 0.05).Moreover,inhibitory effects of tanshinone ⅡA on the migration and invasion of A375 cells,as well as on the mRNA and protein expression of CXCR7,were significantly enhanced with the increase of tanshinone ⅡA concentrations (all P ＜ 0.05).Conclusion Tanshinone ⅡA can inhibit the migratory and invasive abilities of melanoma A375 cells by down-regulating CXCR7 expression.
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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.
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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.
ABSTRACT
Aim To investigate the anti-tumor effects of FS-108 an Hsp90 inhibitor, on oncogene addicted EBC-1 and A375 cells. Methods SRB assay was performed to investigate cell proliferation. Immunoblot was conducted to investigate the specific proteins. FACS was conducted to test cell cycle distribution and apoptosis. Transwell assay was conducted to investigate cell motility. Results FS-108 significantly suppressed cell proliferation of EBC-1 and A375 cancer cells with IC50 at 25. 53 nmol · L-1 and 30. 02 nmol · L-1 re-spectively. FS-108 treatment triggered the degradation of key client proteins such as c-Met and B-Raf and thereby reduced their downstream AKT and ERK signa-ling pathways. The FACS analysis results demonstrated that FS-108 treatment induced G2/M phase arrest and apoptosis significantly. Furthermore, FS-108 inhibited the migration of EBC-1 and A375 cells. Conclusion As a potent Hsp90 inhibitor, FS-108 can inhibit onco-gene addicted cancer cells proliferation through induc-tion of G2/M phase arrest and apoptosis.