rhBMP2 enhances migration and invasion capacity of human breast cancer cells MCF-7

AIM: To determine the effect of rhBMP2 on the migration of human breast cancer cells MCF-7. METHODS: MCF-7 was induced by rhBMP2 (30 μg/L) for 24 h. Atomic force microscopy (AFM) was used to observe the changes in cell morphology. Cell migration and invasion abilities were assayed by scratch healing and transwell experiments. RESULTS: The formation of lamellipodia and cell polarity together with increased cell length were observed in the cells treated with rhBMP2, whereas lamellipodia of cells in control group were not obvious and the majority of cells tended to be rounder with shorter cell diameter. Compared to control group, scratch healing and transwell experiments showed that the migration and invasion capacity of rhBMP2-induced MCF-7 cells was markedly enhanced. CONCLUSION: rhBMP2 induces human breast cancer cell MCF-7 to present the phenotype of migration and enhances the invasion capacity.     

Effects of furin inhibitor on metastasis of human breast cancer MCF-7 cells

AIM：To investigate the mechanism underlying breast cancer metastasis and to provide theoretical data for studying the pathogenesis of breast cancer onset and development. METHODS：Human breast cancer MCF-7 cells were treated with different concentrations of furin inhibitor α1-PDX for 48 h. Wound healing assay and Transwell assay were applied to detect the migration and invasion abilities of the MCF-7 cells. The expression of cell migration-associated proteins, including membrane-type 1 matrix metalloproteinase (MT1-MMP), vascular endothelial growth factor (VEGF)-C and VEGF-D, was determined by Western blotting. The protein levels of MMP2 and MMP9 in the supernatant were measured by ELISA. RESULTS：Compared with control group, 200 nmol/L of furin inhibitor exerted significant inhibitory effects on the cell migration (P<0.05). The expression of cell migration-associated proteins MT1-MMP, VEGF-C and VEGF-D was significantly inhibited after treated with α1-PDX (P<0.05). Significant inhibitory effects of α1-PDX on the expression of MMP9 and MMP2 (P<0.05) in the supernatant were observed. CONCLUSION：Furin inhibitor suppresses the metastasis of MCF-7 cells via down-regulating the expression of MMPs and VEGFs.     

Effects of interferon-inducible protein 16 on proliferation and migration of human brain vascular adventitial fibroblasts

AIM：To investigate the effects of interferon-inducible protein 16 (IFI16) on the proliferation and migration of human brain vascular adventitial fibroblasts (HBVAFs). METHODS：The siRNA of IFI16 gene was transfected into HBVAFs. Forty-eight hours after transfection, the cells were exposed to 2×106 U/L interferon alpha (IFN-α) for 24 h. Cell cycle was analyzed by flow cytometry. Cell migration was determined by scratch assay and transwell method. The mRNA and protein levels of IFI16, p53 and p21 were measured by real-time PCR and Western blotting, respectively. RESULTS：After transfection with IFI16 siRNA, the expression of IFI16, p53 and p21 at mRNA and protein levels was decreased in HBVAFs, and the cell cycle at G1/S transition was promoted. Meanwhile, stimulated with IFN-α up-regulated the expression of IFI16, p53 and p21 at mRNA and protein levels, and inhibited the cell cycle transition at G1/S and cell migration in HBVAFs. Such effect was restrained by transfection with IFI16 siRNA into HBVAFs. CONCLUSION：The expression of IFI16 inhibits the proliferation and migration of HBVAFs, which may be related to the activation of p53 and p21 expression.     

Knockdown of RRM2 gene by siRNA inhibits human breast cancer cell proliferation,migration and tumor growth in nude mice

AIM: To explore the effect of ribonucleotide reductase M2 (RRM2) gene knockdown by siRNA on the proliferation and migration of human breast cancer MCF-7 cells and the tumor growth in BALB/c nude mice. METHODS: The mRNA and protein expression leves of RRM2 in human breast cancer cell line MCF-7 and human normal breast cell line MCF-10A were determined by real-time PCR and Western blotting. siRNA-RRM2 was constructed and transfected into MCF-7 cells at different time points and different concentrations. The silencing efficiency of RRM2 gene was detected by real-time PCR. The cell proliferation was measured by CCK-8 assay. The migration was observed using Transwell cell migration system. The effect of siRNA-RRM2 on the tumor growth was determined in nude mice. RESULTS: The mRNA and protein levels of RRM2 were higher in MCF-7 cells than those in MCF-10A cells. siRNA-RRM2 down-regulated the expression of RRM2 in MCF-7 cells in a time-and concentration-dependent manner. The results of CCK-8 assay showed that siRNA-RRM2 inhibited the proliferation ability of MCF-7 cells, but not that of MCF-10A cells. The results of Transwell assay indicated that siRNA-RRM2 inhibited the migration ability of MCF-7 cells. siRNA-RRM2 also inhibited the tumor growth in nude mice. CONCLUSION: RRM2 overexpression is associated with the breast cancer proliferation and migration. Suppression of RRM2 function is a potential therapeutic strategy for treating breast cancer.     

Effect of digoxin on hypoxia-induced epithelial-mesenchymal transition and invasion in human breast carcinoma MCF-7 cells

AIM: To investigate the effect of digoxin on hypoxia-induced epithelial-mesenchymal transition (EMT), migration and invasion in human breast carcinoma MCF-7 cells. METHODS: MCF-7 cells were treated in vitro with a chemical hypoxia inducer cobalt chloride (CoCl₂) to imitate hypoxia. Cell migration was observed by wound healing assay, and cell invasion was measured by Transwell invasion assay. The protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and vimentin in MCF-7 cells were detected by Western blot. RESULTS: Digoxin inhibited CoCl₂-induced EMT and reversed the mesenchymal phenotype. CoCl₂ enhanced the abilities of migration and invasion (P<0.01), significantly decreased the expression of E-cadherin and increased the expression of HIF-1α, Snail and vimentin (P<0.01), but these effects were blocked by digoxin. CONCLUSION: Digoxin inhibits CoCl₂-induced EMT and invasion most likely via HIF1-α-Snail signaling pathway.     

Effects of isorhapontigenin on cell cycle arrest and down-regulation of cyclin D1 expression in bladder cancer cells

AIM：To explore the inhibitory mechanism of isorhapontigenin (ISO) on the proliferation, migration and invasion of UMUC3 bladder cancer cells. METHODS：Human UMUC3 bladder cancer cells were pretreated with ISO, and the proliferation of the cells was observed under phase-contrast microscope and by ATPase assay. The expression of cyclin D1 was determined by RT-PCR and Western blotting. The cell cycle alteration was detected by flow cytometry, and the cell migration was examined by wound-healing assay. RESULTS：Over 20 μmol/L of ISO significantly inhibited the proliferation of UMUC3 cells with the IC50 of (22.5±2.8) μmol/L. The mRNA and protein levels of cyclin D1 in UMUC3 cells were markedly decreased after treatment with ISO. Exposure of UMUC3 cells to low dose (5 μmol/L) of ISO led to significant induction of G0/G1 growth arrest at both 12 h (58.82%) and 24 h (63.94%), compared with the negative control cells (47.33%) without inducing obvious apoptosis. ISO at dose of 5 μmol/L also markedly inhibited the cell migration. CONCLUSION：ISO significantly exhibits inhibitory effects on the proliferation and migration of human bladder cancer cells by down-regulation of cyclin D1 expression accompanying with G0/G1 cell cycle arrest.     

Baicalein inhibits proliferation and migration of gastric cancer MGC-803 cells

AIM: To investigate the effects of baicalein (BAI) on the proliferation and migration of gastric cancer MGC-803 cells and the mechanisms. METHODS: After MGC-803 cells were treated with BAI at different concentrations, the viability of the MGC-803 cells was tested by MTT assay. The cell colony formation ability were detected by plate colony formation assay. Wound-healing and Transwell cell migration assays were used to test the migration ability of the MGC-803 cells. The concentration of 12-hydroxyeicosatetraenoic acid (12-HETE) was measured by ELISA. The protein levels of platelet type 12-lipoxygenase (p12-LOX), vascular endothelial growth factor (VEGF), p-ezrin and epithelial-mesenchymal transition (EMT) markers in MGC-803 cells were determined by Western blot. RESULTS: BAI significantly inhibited the proliferation, plate colony formation and migration abilities of the MGC-803 cells (P<0.05 or P<0.01), down-regulated the concentration of p12-LOX metabolite 12-HETE significantly (P<0.05 or P<0.01), decreased the protein levels of p12-LOX, VEGF, p-ezrin, vimentin and Snail (P<0.05 or P<0.01), and increased the protein expression of E-cadherin (P<0.01). CONCLUSION: BAI suppresses the proliferation and migration abilities of gastric cancer MGC-803 cells effectively. These effects of BAI may be related to regulating the protein levels of p12-LOX, VEGF, p-ezrin and EMT-related proteins.     

Effects of propofol on expression of CXCR4/CXCR7 and migration ability of human breast cancer MCF-7 cells in vitro

AIM:To investigate the effects of propofol on the expression of CXC-chemokine receptor (CXCR)4/CXCR7 and migration ability of human breast cancer MCF-7 cells in vitro. METHODS:MCF-7 cells were randomly divided into 4 groups,control group, lipid emulsion group, 3 mg/L and 8 mg/L propofol group. The cell viability was measured by MTT assay. The migration ability was detected by wound-healing assay and Transwell assay. The mRNA level of CXCR4/CXCR7 was detected by RT-qPCR. The protein expression leve of CXCR4/CXCR7 was determined by Western blot. RESULTS:Compared with control group, the scratching healing rates in 3 and 8 mg/L propofol group were decreased (P<0.05), and the chemotactic index was also decreased (P<0.05). The protein expression level of CXCR4/CXCR7 was decreased in 3 and 8 mg/L propofol group(P<0.05). However, both the mRNA level of CXCR4/CXCR7 and the viability of the MCF-7 cells kept no change. CONCLUSION:Propofol down-regulates the protein expression of CXCR4/CXCR7 and inhibits the migration ability of breast cancer MCF-7 cells in vitro.     

Mechanism of AKT1 negatively regulating breast cancer cell migration

AIM: To investigate the molecular mechanism and downstream signaling pathway by which AKT1 inhibition regulates breast cancer cell migration. METHODS: RNA interference was used to knockdown the expression of AKT1. Western blot assay was performed to examine the expression of AKT1 total protein, β-catenin total protein and β-catenin nuclear protein. Immunofluorescence was used to examine the cellular localization of β-catenin. Transwell assay was used to investigate whether β-catenin nuclear accumulation as an alternative pathway was responsible for breast cancer metastasis induced by AKT1 inhibition. RESULTS: The total protein expression of AKT1 was decreased in MCF-7 and MDA-MB-231 cells treated with AKT1 siRNA. A significant increase in the protein expression of β-catenin was observed in MCF-7 cells and MDA-MB-231 cells treated with AKT1 siRNA. Immunofluorescence staining showed that MCF-7 cells and MDA-MB-231 cells displayed strong β-catenin staining in the cytoplasm and nucleus after knockdown of AKT1 expression. The ability of tumor cell migration increased dramatically after treated with AKT1 specific siRNA in the breast cancer MCF-7 cells and MDA-MB-231 cells in Transwell assay. XAV-939 reversed breast cancer cell migration induced by knockdown of AKT1 expression. CONCLUSION: β-catenin nuclear accumulation contributes to AKT1 inhibition-mediated breast cancer cell migration.     

Emodin inhibits proliferation and blocks cell cycle of human breast cancer MCF-7 cells

AIM：To investigate the effects of emodin on the proliferation of human breast cancer MCF-7 cells and its mechanisms. METHODS：MTT assay was used to observe the viability of MCF-7 cells. The cell cycle distribution and apoptosis of MCF-7 cells was analyzed by flow cytometry. The membrane surface morphology and three-dimensional ultrastructure of MCF-7 cells were observed under atomic force microscope (AFM). RESULTS：MTT assay showed that emodin could inhibit MCF-7 cell proliferation in a dose-dependent manner. Flow cytometric analysis demonstrated that emodin induced cell cycle arrest at G0/G1 phase. Annexin V/PI double staining confirmed that emodin had no effect on cell apoptosis. AFM images revealed that the cell nuclear area was full and the surface of cell membrane was flat and smooth in control group. Compared with control group, the cell nuclear area collapsed and shrank in emodin group at 48 h. The cell membrane ultrastructure showed that the particles in emo-din group had an intensive distribution. The height of cell nuclear area was decreased, and the surface average roughness (Ra) and root mean square roughness (Rq) were elevated in emo-din group compared with control group. CONCLUSION: Emodin has cytotoxicity on MCF-7 cells via cell cycle arrest at G0/G1 phase and ultrastructural changes.     

Effect of IGFBP7 on proliferation of human breast cancer cell line MCF-7

AIM: To investigate the mechanism that insulin-like growth factor binding protein 7 (IGFBP7) inhibits proliferation of human breast cancer cell line MCF-7. METHODS: Plasmid pCMV6-IGFBP7 or empty plasmid was transfected into MCF-7 cells. The expression of IGFBP7 in MCF-7 cells after transfection was detected by Western blotting. The effects of IGFBP7 on the colony-forming efficiency and the cell cycle were studied by soft agar colony formation assay and flow cytometry,respectively. The effects of IGFBP7 on the expression of ERK1/2, p-ERK1/2, cyclin D1, CDK4, cyclin E, CDK2, p21^CIP1/WAF1, p27^KIP1, p53, Rb and p-Rb in MCF-7 cells were detected by Western blotting. RESULTS: Only the transfectant of pCMV6-IGFBP7 expressed IGFBP7. IGFBP7 remarkably reduced colony-forming efficiency (P<0.01) and G₀/G₁ arrest (P<0.01), inhibited phosphorylation of ERK1/2 (P<0.01), down-regulated cyclin D1 and cyclin E (P<0.01), up-regulated p27^KIP1, p21^CIP1/WAF1 and p53 (P<0.01), and inhibited phosphorylation of Rb (P<0.01) in MCF-7 cells. PD98059, an inhibitor of MEK1 and MEK2, imitated part of the tumor-suppressing activity of IGFBP7. CONCLUSION: IGFBP7 inhibits the proliferation of human breast cancer cell line MCF-7 by down-regulating cyclin D1 and cyclin E, up-regulating p27^KIP1, p21^CIP1/WAF1 and p53 and inhibiting phosphorylation of Rb. ERK1/2 signaling pathway might be involved in the regulation of cyclin D1 and p27^KIP1 by IGFBP7.     

Inhibitory effect of HET0016 on LPS-induced proliferation and migration of microglia

AIM To study the effects of HET0016 on the proliferation and migration of microglia stimulated by lipopolysaccharide (LPS). METHODS Primary microglia from neonatal SD rats were isolated, purified and cultured. CCK-8 assay was performed to detect the effect of HET0016 on the viability of microglia after treatment with LPS. The levels of 20-hydroxyeicosatetraenoic acid （20-HETE）, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured by ELISA. The proportion of S phase was evaluated by flow cytometry. The cell migration ability was detected by Transwell assay and scratch wound healing assay. The protein expression of NF-κB p50 and p65 was determined by Western blot. RESULTS LPS induced the increases in the proliferation and migration of microglia and the release of inflammatory cytokines (P<0.05). Compared with LPS group, HET0016 inhibited the cell proliferation and migration (P<0.05), decreased the levels of TNF-α and IL-1β (P<0.05), and reduced the expression of NF-κB p50 and p65 (P<0.05). CONCLUSION HET0016 has inhibitory effects on the proliferation and migration of microglia induced by LPS, and reduces the release of inflammatory cytokines. The mechanism may be related to NF-κB signaling pathway.     

Effect of hirsutine on hypoxia-induced migration and invasion abilities in human breast cancer MCF-7 cells

AIM: To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS: CCK-8 assay was employed to detect the cytotoxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay, and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS: Hirsutine remarkably reduced the cell viability from 32 μmol/L (P<0.05), and the IC₅₀ value was 62.82 μmol/L. In hypoxia state, MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α, Snail and MMP-9 (P<0.05), lower protein level of E-cadherin (P<0.05), and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05), apart from the mRNA level of HIF-1α. CONCLUSION: Hirsutine inhibits hypoxia-induced migration and invasion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α, Snail and MMP-9, and up-regulation of the protein level of E-cadherin.     

3-MA combined with TSA inhibits growth of triple-negative breast cancer cells

AIM: To investigate the effect of 3-methyladenine (3-MA) combined with trichostatin A (TSA) on triple-negative breast cancer cells and the mechanism involved. METHODS: The viability of MDA-MB-231 cells was detected by MTT assay, the migration ability was determined by scratch assay, and the expression of autophagy-related proteins was detected by Western blot. RESULTS: TSA significantly inhibited the viability of MDA-MB-231 cells in a time-and dose-dependent manner. The results of scratch assay showed that TSA inhibited cell migration ability. Western blot data indicated that TSA resulted in a moderate increase in LC3-Ⅱ expression. Moreover, 3-MA inhibited cell autophagy induced by TSA, and combination of 3-MA and TSA further inhibited the viability of MDA-MB-231 cells. CONCLUSION: Combination of 3-MA and TSA may effectively inhibit the growth of triple-negative breast cancer cells.     

Curcumin inhibits migration and invasion of lung cancer PC-9 cells via down-regulation of nectin-4 expression

AIM: To investigate the effects of curcumin on the abilities of migration and invasion in the lung cancer PC-9 cells, and to observe the relationship between curcumin and nectin-4 expression.METHODS: The viability, migration and invasion of lung cancer PC-9 cells treated with curcumin or transfected with siNectin-4 were measured by MTT assay, wound healing test and Transwell assay, respectively. The protein levels of nectin-4, p-AKT and AKT in the PC-9 cells treated with curcumin or transfected with siNectin-4 were detected by Western blot.RESULTS: Curcumin inhibited the viability of PC-9 cells. The wound healing rates and the numbers of the transmembrane cells in curcumin 10 μmol/L and 20 μmol/L groups were decreased compared with control group without curcumin treatment. The expression level of nectin-4 was reduced after curcumin treatment for 24 h. The viability of the PC-9 cells was significantly inhibited after transfected with siNectin-4 for 48 h or 72 h (P<0.01), and the wound healing rates was decreased in siNectin-4 group compared with NC group (P<0.01). The numbers of the transmembrane cells in siNectin-4 group was significantly reduced (P<0.01). Curcumin and knockdown of nectin-4 suppressed the activation of AKT pathway in PC-9 cells. In siNectin-4+curcumin group, the cell viability reduced compared with curcumin group, and wound healing rates, cell invasive ability and AKT phosphorylation levels were decreased.CONCLUSION: Curcumin inhibits migration and invasion of the lung cancer PC-9 cells via down-regulation of nectin-4 expression and inhibition of AKT pathway.     

Long non-coding RNA PVT1 regulates effect of miR-551 on migration and invasion abilities of ovarian cancer through Wnt signaling pathway

AIM: To investigate the expression of long non-coding RNA PVT1 in ovarian cancer and the role of PVT1 in migration and invasion abilities of ovarian cancer cells.METHODS: The expression of PVT1 in ovarian cancer tissue, normal ovarian tissue and different ovarian cancer cell lines was detected by qPCR. Transwell assay was used to detect the invasion ability of ovarian cancer cells after PVT1 silencing. The migration ability of the ovarian cancer cells after PVT1 silencing was detected by scratch test. The interaction between PVT1 and microRNA (miR)-551 was analyzed by dual-luciferase reporter assay. The effect of miR-551-inhibitor on the invasion and migration abilities of ovarian cancer cells after PVT1 silencing was detected by Transwell assay and scratch test. The expression of Wnt signaling pathway-related proteins was determined by Western blot after PVT1 silencing. The effects of PVT1 silencing on tumor weight and volume of ovarian cancer were examined by subcutaneous tumor transplantation in nude mice.RESULTS: The expression of PVT1 in ovarian cancer tissue was significantly higher than that in normal ovarian tissue (P<0.05). The expression level of PVT1 in ovarian cancer cell line ES-2 was the highest. PVT1 silencing inhibited the invasion and migration abilities of the ovarian cancer cells. After PVT1 silencing, miR-551-inhibitor promoted the invasion and migration abilities of the ovarian cancer cells. The expression of Wnt signaling pathway-related proteins was decreased after PVT1 silencing (P<0.05). Compared with negative control group, the tumor volume and weight in PVT1-siRNA group were significantly decreased (P<0.05).CONCLUSION: PVT1 plays an important role in the development of ovarian cancer. PVT1 regulates the invasion and migration abilities of ovarian cancer cells through Wnt signaling pathway.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

Role of IGF-Ⅱ in proliferation and migration of hepatocellular carcinoma Huh7 cells

AIM: To observe the effects of insulin-like growth factorⅡ (IGF-Ⅱ) at different expression levels on hepatocellular carcinoma Huh7 cell proliferation and migration, and to explore the role of IGF-Ⅱ in the development of HCC. METHODS: The Huh7 cells were transfected with the over-expression plasmid pcDNA3.1(+)- IGF-Ⅱ or RNA interference plasmid pLVX-shRNA-IGF-Ⅱ by Lipofectamine 2000. The quantitative real-time PCR and Western blotting were used to verify the expression of IGF-II. The biological behaviors of the Huh7 cells were analyzed by CCK-8 assay, plate clone formation assay, cell scratch test and Transwell chamber experiment.RESULTS: Over-expression of IGF-II promoted the growth and migration of hepatocellular carcinoma cells (P < 0.05), and the cell proliferation was significantly inhibited in the Huh7 cells with low IGF-II expression (P < 0.05).CONCLUSION: IGF-II is involved in the regulation of biological behavior of hepatocellular carcinoma Huh7 cells in vitro, which may play a promoting role in the development of hepatocellular carcinoma.