MAGED4B belongs to the melanoma-associated antigen family; originally found in melanoma, it is expressed in various types of cancer, and is especially enriched in glioblastoma. However, the functional role and molecular mechanisms of MAGED4B in glioma are still unclear. In this study, we found that the MAGED4B level was higher in glioma tissue than that in non-cancer tissue, and the level was positively correlated with glioma grade, tumor diameter, Ki-67 level, and patient age. The patients with higher levels had a worse prognosis than those with lower MAGED4B levels. In glioma cells, MAGED4B overexpression promoted proliferation, invasion, and migration, as well as decreasing apoptosis and the chemosensitivity to cisplatin and temozolomide. On the contrary, MAGED4B knockdown in glioma cells inhibited proliferation, invasion, and migration, as well as increasing apoptosis and the chemosensitivity to cisplatin and temozolomide. MAGED4B knockdown also inhibited the growth of gliomas implanted into the rat brain. The interaction between MAGED4B and tripartite motif-containing 27 (TRIM27) in glioma cells was detected by co-immunoprecipitation assay, which showed that MAGED4B was co-localized with TRIM27. In addition, MAGED4B overexpression down-regulated the TRIM27 protein level, and this was blocked by carbobenzoxyl-L-leucyl-L-leucyl-L-leucine (MG132), an inhibitor of the proteasome. On the contrary, MAGED4B knockdown up-regulated the TRIM27 level. Furthermore, MAGED4B overexpression increased TRIM27 ubiquitination in the presence of MG132. Accordingly, MAGED4B down-regulated the protein levels of genes downstream of ubiquitin-specific protease 7 (USP7) involved in the tumor necrosis factor-alpha (TNF-α)-induced apoptotic pathway. These findings indicate that MAGED4B promotes glioma growth via a TRIM27/USP7/receptor-interacting serine/threonine-protein kinase 1 (RIP1)-dependent TNF-α-induced apoptotic pathway, which suggests that MAGED4B is a potential target for glioma diagnosis and treatment.
Humans ; Tumor Necrosis Factor-alpha ; DNA-Binding Proteins/metabolism* ; Ubiquitin-Specific Peptidase 7 ; Cisplatin ; Temozolomide ; Transcription Factors ; Glioma ; Cell Proliferation ; Melanoma ; Cell Line, Tumor ; Apoptosis ; Nuclear Proteins/genetics*

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

Melanoma is the most aggressive cutaneous tumor. Neuropilin and tolloid-like 2 (NETO2) is closely related to tumorigenesis. However, the functional significance of NETO2 in melanoma progression remains unclear. Herein, we found that NETO2 expression was augmented in melanoma clinical tissues and associated with poor prognosis in melanoma patients. Disrupting NETO2 expression markedly inhibited melanoma proliferation, malignant growth, migration, and invasion by downregulating the levels of calcium ions (Ca²⁺) and the expression of key genes involved in the calcium signaling pathway. By contrast, NETO2 overexpression had the opposite effects. Importantly, pharmacological inhibition of CaMKII/CREB activity with the CaMKII inhibitor KN93 suppressed NETO2-induced proliferation and melanoma metastasis. Overall, this study uncovered the crucial role of NETO2-mediated regulation in melanoma progression, indicating that targeting NETO2 may effectively improve melanoma treatment.
Humans ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Melanoma/genetics* ; Membrane Proteins/genetics* ; Phosphorylation ; Signal Transduction

Catechins have been proven to exert antitumor effects in different kinds of cancers. However, the underlying mechanisms have not been completely clarified yet. This study aimed to assess the effects and mechanisms of (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) on human melanoma skin A375 cells. Results showed that EGCG and ECG inhibited the proliferation of A375 cells and ECG showed better inhibitory effect. Flow cytometry analysis had shown that EGCG and ECG induced apoptosis and led to cell cycle arrest. EGCG and ECG decreased Bcl-2 expression and upregulated Caspase-3 protein level, indicating the development of apoptosis. Furthermore, EGCG and ECG could decreased mitochondrial membrane potential of A375 cells. In addition, the expression of Beclin-1, LC3 and Sirt3 were downregulated at protein levels, which known to be associated with autophagy. After autophagy was increased by rapamycin, the apoptotic trend was not change, indicating that apoptosis and autophagy are independent. Mechanistically, EGCG and ECG treatments decreased phosphorylated-AMPK (p-AMPK) and increased the ratios of p-PI3K, p-AKT and p-mTOR in melanoma cells. Conclusively, EGCG and ECG induced apoptosis via mitochondrial signaling pathway, downregulated autophagy through modulating the AMPK/mTOR and PI3K/AKT/mTOR signaling pathway. It indicated that EGCG and ECG may be utilized in human melanoma treatment.
AMP-Activated Protein Kinases/genetics* ; Apoptosis ; Autophagy ; Catechin/analogs & derivatives* ; Electrocardiography ; Humans ; Melanoma/drug therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVES: To investigate the effects of injury time, postmortem interval (PMI) and postmortem storage temperature on mRNA expression of glycoprotein non-metastatic melanoma protein B (Gpnmb), and to establish a linear regression model between Gpnmb mRNA expression and injury time, to provide aimed at providing potential indexes for injury time estimation. METHODS: Test group SD rats were anesthetized and subjected to blunt contusion and randomly divided into 0 h, 4 h, 8 h, 12 h, 16 h, 20 h and 24 h groups after injury, with 18 rats in each group. After cervical dislocation, 6 rats in each group were collected and stored at 0 ℃, 16 ℃ and 26 ℃, respectively. The muscle tissue samples of quadriceps femoris injury were collected at 0 h, 12 h and 24 h postmortem at the same temperature. The grouping method and treatment method of the rats in the validation group were the same as above. The expression of Gpnmb mRNA in rat skeletal muscle was detected by RT-qPCR. The Pearson correlation coefficient was used to evaluate the correlation between Gpnmb mRNA expression and injury time, PMI, and postmortem storage temperature. SPSS 25.0 software was used to construct a linear regression model, and the validation group data was used for the back-substitution test. RESULTS: The expression of Gpnmb mRNA continued to increase with the prolongation of injury time, and the expression level was highly correlated with injury time (P<0.05), but had little correlation with PMI and postmortem storage temperature (P>0.05). The linear regression equation between injury time (y) and Gpnmb mRNA relative expression (x) was y=0.611 x+4.489. The back-substitution test proved that the prediction of the model was accurate. CONCLUSIONS The expression of Gpnmb mRNA is almost not affected by the PMI and postmortem storage temperature, but is mainly related to the time of injury. Therefore, a linear regression model can be established to infer the time of injury.
Animals ; Rats ; Glycoproteins ; Linear Models ; Melanoma ; Membrane Glycoproteins/genetics* ; Postmortem Changes ; Rats, Sprague-Dawley ; RNA, Messenger/metabolism* ; Time Factors

Increasing evidence suggests that stress may induce changes in hair color, with the underlying mechanism incompletely understood. In this study, female C57BL/6 mice subjected to electric foot shock combined with restraint stress were used to build chronic stress mouse model. The melanin contents and tyrosinase activity were measured in mouse skin and B16F10 melanoma cells. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor α (TNF-α), interleukin- 1β (IL-1β) and interleukin-6 (IL-6) in the mouse skin. The content of nuclear factor κB (NFκB)/p65 subunit in mouse skins was valued by immunofluorescence staining. The results demonstrated that under chronic stress, the fur color turned from dark to brown in C57BL/6 mice due to the decrease of follicle melanocytes and tyrosinase activity in C57BL/6 mouse skin. Simultaneously, inflammatory responses in skins were detected as shown by increased NFκB activity and TNF-α expression in stressed mouse skin. In cultured B16F10 melanoma cells, TNF-α reduced the melanogenesis and tyrosinase activity in a dose-dependent manner. These findings indicate that chronic stress induces fur color change by decreasing follicle melanocytes and tyrosinase activity in female C57BL/6 mice, and TNF-α may play an important role in stress-induced hair color change.
Animal Fur ; Animals ; Color ; Female ; Melanins ; Melanocytes ; enzymology ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; Monophenol Monooxygenase ; metabolism ; Pigmentation ; Skin ; physiopathology ; Stress, Physiological

To study the effects of the anthocyanin single component cyanidin-3-O-glucoside (Cy-3-glu) on the proliferation and migration of mouse melanoma cells and to elucidate the underlying mechanisms, B16-F10 cells were treated with different concentrations of Cy-3-glu. Cell viability was analyzed by a CCK-8 method. Cell migration was determined by the callus scratching technique. Cell cycle was measured by the flow cytometry. The expression levels of genes involved in cell cycle regulation were detected by real-time PCR. Protein expression levels of p-AKT, E-cadherin, N-cadherin and vimentin were analyzed by Western blot. The growth and migration of B16-F10 cells in C57BL/6J mice were monitored by the cryogenically cooled IVIS-imaging system. The results showed that Cy-3-glu significantly inhibited the growth (P < 0.001) and migration (P < 0.01) of B16-F10 cells, and arrested the cell cycle in the S phase. After Cy-3-glu treatment, the expression levels of p-AKT (P < 0.05), N-cadherin and vimentin (P < 0.001) were decreased significantly, and the expression level of E-cadherin was dramatically increased (P < 0.05). The size and weight of tumors and tumor metastasis in mice fed with a diet containing Cy-3-glu were significantly reduced (P < 0.05). In conclusion, Cy-3-glu inhibits proliferation and migration of B16-F10 cells by inhibiting the PI3K/AKT signaling pathway, cell adhesion and migration signals.
Animals ; Anthocyanins ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Glucosides ; pharmacology ; Melanoma, Experimental ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases ; metabolism

OBJECTIVE: To investigate the effects of millimeter wave (MMW) exposure on apoptosis of human melanoma A375 cells and explore the mechanisms. METHODS: Through electromagnetic field calculation we simulated MMW exposure in cells and calculated the specific absorption rate (SAR). The optimal irradiation parameters were determined according to the uniformity and intensity of the SAR. A375 cells were then exposed to MMV for 15, 30, 60, or 90 min, with or without pretreatment with the caspase-3 inhibitor AC-DEVD-fmk (10 μmol/L) for 1 h at 90 min before the exposure. CCK-8 assay was used to assess the changes in the viability and Annexin-V/ PI staining was used to detect the apoptosis of the cells following the exposures; Western blotting was used to detect the expression of caspase-3 in the cells. RESULTS: The results of electromagnetic field calculation showed that for optimal MMV exposure, the incident field needed to be perpendicular to the bottom of the plastic Petri dish with the antenna placed below the dish. CCk-8 assay showed that MMW exposure significantly inhibited the cell viability in a time-dependent manner ( < 0.05); exposures for 15, 30, 60, and 90 min all resulted in significantly increased apoptosis of the cells ( < 0.05). The cells with MMW exposure showed significantly increased expression of caspase-3. The inhibitory effect of MMW on the cell viability was antagonized significantly by pretreatment of the cells with AC-DEVD-fmk ( < 0.05), which increased the cell viability rate from (36.7±0.09)% to (59.8±0.06)% ( < 0.05). CONCLUSIONS 35.2 GHz millimeter wave irradiation induces apoptosis in A375 cells by activating the caspase-3 protein.
Apoptosis ; Caspase 3 ; metabolism ; Caspase Inhibitors ; pharmacology ; Cell Line, Tumor ; Cell Survival ; Electromagnetic Fields ; Enzyme Activation ; Humans ; Magnetic Field Therapy ; Melanoma ; enzymology ; pathology ; therapy ; Time Factors

OBJECTIVE: To investigate the expression profile of miR-122-5p in melanoma tissues and the effect of miR-122-5p on the proliferation, cell cycle and apoptosis of human melanoma cell lines SK-MEL-110 and A375. METHODS: The expression profiles of miR-122-5p in melanoma and pigmented nevus tissues were detected using real-time fluorescence quantitative PCR (qRT-PCR). SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor or negative control inhibitor (NC) I were examined for miR-122- 5p expression using qRT-PCR and changes in cell proliferation, cell cycle and apoptosis using MTT assay or flow cytometry. NOP14 mRNA and protein expressions in the cells were detected using qRT- PCR and Western blotting, respectively. Luciferase reporter assay was used to confirm the identity of NOP14 as the direct target of miR-122-5p. RESULTS: The relative expression of miR-122-5p in human pigmented nevus tissues and melanoma tissues was 1.23±0.270 and 7.65 ± 1.37, respectively. The relative expression of miR-122-5p in SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor was 0.21 ± 0.08 and 0.17 ± 0.05, respectively. miR-122-5p inhibitor obviously inhibited the cell proliferation and increased the percentage of cells in G1 stage in both SK-MEL-110 and A-375 cells, but did not cause obvious changes in the apoptosis of the two cells. miR-122-5p inhibitor did not significantly affect the expression level of NOP14 mRNA, but obviously increased the expression level of NOP14 protein. Luciferase reporter assay revealed a significantly lower luciferase activity in cells co-transfected with miR-122-5p mimics and wild-type psi-CHECK2-3'UTR plasmid than in the cells cotransfected with NC and wild-type psi-CHECK2-3'UTR plasmid (0.21 ± 0.14 0.56 ± 0.1, < 0.01). CONCLUSIONS miR-122-5p expression is upregulated in melanoma tissues, indicating its involvement in the development of melanoma. miR-122-5p inhibits the proliferation of SK-MEL-110 and A-375 cells possibly by affecting the cycle through NOP14.
Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Luciferases ; metabolism ; Melanoma ; etiology ; metabolism ; pathology ; MicroRNAs ; antagonists & inhibitors ; metabolism ; Neoplasm Proteins ; metabolism ; Nevus, Pigmented ; etiology ; metabolism ; pathology ; Nuclear Proteins ; metabolism ; Skin Neoplasms ; etiology ; metabolism ; pathology ; Up-Regulation

OBJECTIVETo detect the expression of protein 4.1 family members in mouse melanoma cell lines and evaluate their effect on cell proliferation.

METHODSPCR and Western blot were used to detected to the expression of protein 4.1 family members (4.1R, 4.1B, 4.1G, and 4.1N) at the mRNA and protein levels in B16 and B16-F10 cell lines. The expression plasmid vector pEGFP-N1-EPB41L3 carrying 4.1B gene sequence amplified from genomic RNA of mouse embryo fibroblasts was constructed and transiently transfected into mouse melanoma cells. The change in cell proliferation was assessed using MTT assay.

RESULTSThe mRNA and protein expressions of all the protein 4.1 family members, with the exception of 4.1B, were detected in both B16 and B16-F10 cells. Transfection of cells with the eukaryotic expression vector pEGFP-N1-EPB41L3 markedly inhibited cell proliferation as compared with the non-transfected cells.

CONCLUSIONThe eukaryotic expression vector carrying EPB41L3 sequence is capable of inhibiting the proliferation of mouse melanoma B16 and B16-F10 cells.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Genetic Vectors ; Melanoma, Experimental ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; Neuropeptides ; metabolism ; Plasmids ; Transfection