CDA-2 induces differentiation and inhibits proliferation of human SWO-38 glioma cells

AIM: To investigate the differentiation-inducing effect of cell differentiation agent-2 (CDA-2) in human SWO-38 glioma cell line in vitro.METHODS: The inhibitory effect of CDA-2 on cell proliferation was assessed by MTT assay and colony formation assay.Cell morphology was determinded by light microscopy observation,and the expression of GFAP (glial fibrillary acidic protein) was detected by immunohistochemistry and Western blotting.Western blotting was also applied to explore the expression of PPARγ and COX-2.RESULTS: The data showed that CDA-2 inhibited proliferation and induced differentiation of SWO-38 cells.The inhibition efficiency was time-dependent and dose-dependent .The IC₅₀ of CDA-2 was (2.33±0.37) g/L and (0.51±0.01) g/L,respectively when cells were treated for 72 h and 10 days.CDA-2 caused differentiation of human glioma cells as indicated by outgrowth of long processes and expression of astrocyte marker GFAP.Simultaneously,the expression of PPARγ increased after 3 h of CDA-2 treatment，while the expression of COX-2 decreased after 48 h of CDA-2 treatment.CONCLUSION: CDA-2 inhibits proliferation and induces differentiation of SWO-38 cells.These effects may be through increasing cellular GFAP,PPARγ level and decreasing COX-2 expression induced by CDA-2.     

Effects of lentivirus-mediated siRNA targeting IGF-1R on migration and invasion abilities of endometrial carcinoma cells

AIM:To investigate the down-regulation of insulin-like growth factor tgpe 1 receptor(IGF-1R) on the migration and invasion abilities of human endometrial cancer cell HEC-1B. METHODS:The siRNAs targeting IGF-1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC-1B cells(HEC-1B-KD group). The control cells(without virus transfection, HEC-1B-CON group) and negative virus transfection control cells(HEC-1B-NC group) were also set up. The gene silencing effect of siRNA targeting IGF-1R was determined by real-time PCR and Western blotting at mRNA and protein levels,respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experiment. The mRNA levels of matrix metalloproteinase(MMP)-2 and MMP-9 were measured by real-time PCR. RESULTS:The mRNA and protein levels of IGF-1R in HEC-1B-KD cells were significantly reduced by 81% and 91.5%, respectively(P<0.05). In anchorage-dependent growth by colony formation assay, HEC-1B-KD cells showed much less colonies than HEC-1B-CON cells and HEC-1B-NC cells. Compared with the control cells, knockdown of IGF-1R in HEC-1B cells resulted in significant reduction of cell motility. Down-regulation of IGF-1R in HEC-1B cells also significantly reduced the invasion potential(P<0.05). Down-regulation of IGF-1R substantially reduced the expression of MMP-2 and MMP-9 compared with the control cells. CONCLUSION:Knockdown of IGF-1R reduces the migration and invasion abilities of human endometrial cancer cells in vitro accompanied with a decrease in MMP-2 and MMP-9 expression.     

Change of nestin expression in differentiation of human glioma cells induced by CDA-2

AIM: To investigate the change and significance of nestin expression in differentiation of human glioma cell line SWO-38 induced by CDA-2 (uroacitide, a healthy human urine extract). METHODS: Cellular differentiation of SWO-38 cells induced by CDA-2 was determined by light microscopy. The change of nestin expression in SWO-38 cells induced by CDA-2 was detected by munofluorescence, RT-PCR and Western blotting. RESULTS: Light microscopic observation revealed that CDA-2 induced SWO-38 cells to differentiate into astrocytes with increased cytoplasm and cytoplasmic processes and decreased nucleus/cytoplasm ratio. Nestin was expressed in cytoplasm and stained like filament by immunofluorescence staining. Nestin expression was downregulated in differentiated SWO-38 cells induced by CDA-2. CONCLUSION: Nestin expression is downregulated in differentiation of SWO-38 cells induced by CDA-2, which verifies the relationship between nestin expression and cell differentiation. Nestin may be a new differentiation marker of glioma.     

Effects of propofol on invasion and migration of human gastric cancer SGC-7901 cells

AIM: To investigate the effects of propofol on invasion and migration of gastric cancer cell line SGC-7901. METHODS: Cultured gastric cancer cell line SGC-7901 was randomly divided into 4 groups, and then diffe-rent concentrations (1, 3, 5 and 7 mg/L) of propofol were added and incubated for 24 h. The cell viability was measured by MTT assay. The invasion and migration abilities of the SGC-7901 cells were detected by Transwell assay and wound-healing assay. The expression of cysteine-rich angiogenic inducer 61 (CYR61), CD44v6 and matrix metalloproteinase-7 (MMP-7) in the SGC-7901 cells were examined by immunocytochemistry and Western blot.RESULTS: Propofol at 5 mg/L does not affect the viability of SGC-7901 cells, whereas significantly suppresses the invasion and migration abilities, and down-regulates the expression of CD44v6 and MMP-7 (P<0.05). CONCLUSION: The decreased invasion and migration abilities of SGC-7901 cells were partly due to the inhibition of CD44v6 and MMP-7 expression.     

Expression of Gab2 in human osteosarcoma U2-OS cells

AIM: To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteosarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells. METHODS: The technique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines. Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells. After transfection, through chemotaxis and invasion assays in vitro, the cell migration and invasion abilities were detected. RESULTS: After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells (SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells (Scr/U2-OS) and U2-OS cells. After stimulation with epidermal growth factor (EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells (P<0.01). The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups (P<0.01). CONCLUSION: Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.     

Effects of SM22α on migration and invasion abilities of colorectal cancer cells

AIM:To explore the effects of smooth muscle 22α protein (SM22α) on the migration and invasion abilities of colorectal cancer cells, and to investigate its molecular mechanism. METHODS:The SM22α-over-expressing cells were constructed by lentivirus transfection. The cell migration ability was detected by wound healing assay. The changes of cell migration and invasion abilities were measured by Transwell assay. RT-qPCR was used to detect the changes of SM22α mRNA level. The protein levels of extracellular signal-regulated kinase (ERK), p-ERK, matrix metalloproteinase-9 (MMP-9) and SM22α were determined by Western blot. RESULTS:HCT116 cells with SM22α over-expression were constructed successfully. SM22α inhibited the migration and invasion abilities of colorectal cancer cells. SM22α over-expression decreased the protein levels of p-ERK and MMP-9 (P<0.05).CONCLUSION:SM22α inhibits the migration and invasion abilities of colorectal cancer cells by inhibiting ERK/MMP-9 signaling pathways.     

Knockdown of Notch-1 augments inhibitory effect of dihydroartemisinin on viability of human osteosarcoma cell line U-2OS

AIM: To investigate the effect of Notch-1 knockdown on the growth of dihydroartemisinin-inhibited human osteosarcoma cell line U-2OS. METHODS: U-2OS cells treated with different concentrations of dihydroartemisinin (5, 10, 15 and 20 μmol/L) were collected. The expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively. U-2OS cells were transfected with Notch-1 siRNA for 24 h and incubated with dihydroartemisinin for another 24 h. The cell apoptotic rate, protein expression of MMP-2, MMP-9 and Hes-1, and the migration ability were measured by MTT assay, Western blotting and Transwell experiment, respectively. RESULTS: Dihydroartemisinin (5, 10, 15 and 20 μmol/L) decreased the expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels in a dose-dependent manner. Down-regulation of Notch-1 significantly enhanced the effect of dihydroartemisinin on the cell apoptosis, the protein expression of MMP-2, MMP-9 and Hes-1, and migration ability (P<0.05). CONCLUSION: Notch-1 pathway is involved in the process of dihydroartemisinin-inhibited U-2OS cell growth. Knockdown of Notch-1 augments the inhibitory effect of dihydroartemisinin on U-2OS cell viability.     

Effects of TREM-2 silencing on migration and invasion abilities of rheumatoid arthritis fibroblast-like synoviocytes

AIM: To investigate the effects of triggering receptor expressed on myeloid cells-2 (TREM-2) silencing on migration and invasion abilities of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). METHODS: Small interference RNA (siRNA) specifically targeting TREM-2 gene was transfected into RA-FLS. The interference efficiency of TREM-2 siRNA on the production of TREM-2 mRNA and protein was determined by RT-PCR and Western blot. The cell activity was assessed by CCK-8 assay. The migration and invasion abilities of RA-FLS were determined by Transwell assay. The releases of MMP-2 and MMP-9 in RA-FLS were analyzed by ELISA. The influence of TREM-2 on PI3K/AKT signal pathway was measured by Western blot. RESULTS: TREM-2 siRNA significantly decreased the mRNA and protein expression of TREM-2. No difference of cell activity between TREM-2 siRNA group and control group was observed. Transwell migration assay showed that RA-FLS through the Transwell membrane in TREM-2 siRNA group were more than the blank control group and the NC-siRNA group. In Transwell invasion assay, RA-FLS through the Transwell membrane in TREM-2 siRNA group were more than the blank control group and the NC-siRNA group. After transfected with TREM-2 siRNA, the MMP-2 secretion and phosphorylation of AKT increased significantly, while the MMP-9 secretion was not changed. CONCLUSION: TREM-2 may play an important role in the migration and invasion of RA-FLS through regulating the activation of PI3K/AKT signal pathway.     

Overexpression of HBXIP in HepG2 cells induces cell migration and regulates expression of β-catenin

AIM: To investigate the effects of hepatitis B virus X-interacting protein(HBXIP) in hepatic cancer cells on the cell migration and expression of β-catenin. METHODS: Transwell assay was used to assess the cell migration. Gelatin zymography was used to observe the activity of matrix metalloproteinase 9 (MMP-9). The expression of MMP-9, glycogen synthase kinase 3β(GSK-3β), p-GSK3β, β-catenin and p-β-catenin in HepG2 cells was determined by Western blotting. RESULTS: HepG2 cells which stably overexpressed HBXIP (HepG2-HBXIP) exhibited higher migration ability than the control cells. The results of the gelatin zymography assay showed that HBXIP overexpression increased the activity of MMP-9 in HepG2 cells. The results of Western blotting indicated that HBXIP increased the expression of MMP-9 and β-catenin, inhibited the phosphorylation of β-catenin and promoted the phosphorylation of GSK-3β (Ser9). CONCLUSION: HBXIP regulates the GSK-3β/β-catenin signaling pathway, resulting in a significant improvement of hepatocellular carcinoma cell migration.     

Effects of axitinib on biological behavior of adrenocortical carcinoma cell line SW-13

AIM: To investigate the effects of axitinib on the biological behavior of adrenocortical carcinoma cell line SW-13. METHODS: CCK-8 assay was used to measured the viability of SW-13 cells treated with axitinib at different concentrations. The cell cycle distribution was analyzed by flow cytometry. The apoptotic rate was also analyzed by flow cytometry with Annexin V/PI double staining. Wound healing experiment and Transwell invasion assay were used to observe cell migration and invasion abilities,respectively. The protein levels of vascular endothelial growth factor receptor 2(VEGFR2), extracellular regulated protein kinases 1/2 (ERK1/2) and p-ERK1/2 were determined by Western blot. RESULTS: After treated with axitinib, the viability of SW-13 cells was significantly inhibited, the cell cycle was blocked in G₂/M phase, and the apoptosis rate was increased. The migration and invasion abilities of SW-13 cells were markedly inhibited by axitinib (P<0.01). The protein levels of VEGFR2 and p-ERK1/2 in the SW-13 cells were significantly decreased with axitinib treatment (P<0.01). CONCLUSION: Axitinib inhibits the viability, blocks the cell cycle, promotes cell apoptosis, and inhibits the migration and invasion abilities of SW-13 cells. The mechanism may be related to inhibition of VEGFR2 expression and reduction of ERK1/2 phosphorylation.     

Chronic hypoxia enhances aggressiveness of MCF-7 breast cancer cells through EMT

AIM: To investigate the effects of chronic hypoxia on the aggressiveness of MCF-7, a human breast cancer cell line, and the underlying mechanisms.METHODS: MCF-7 cells were cultured under hypoxia (1% O₂, 5% CO₂ and 94% N₂) or control (95% O₂ and 5% CO₂) condition. The viability, proliferation, and invasion and migration abilities of the MCF-7 cells were determined by MTT assay, CCK-8 assay, cell counting, and cell invasion and migration assays. Anchorage-independent growth and the alteration of cellular polarization of the MCF-7 cells were tested by soft agar colony formation assay and Matrigel-3D culture assay, respectively. The effects of chronic hypoxia on the growth and metastasis of MCF-7 cells in vivo were investigated by xenograft in nude mice. The morphological changes of the MCF-7 cells were observed under an inverted microscope. Hypoxia-induced alterations in the levels of hypoxia inducible factor-1 (HIF-1) and phosphorylated glycogen synthase kinase-3β (p-GSK-3β) as well as epithelial-mesenchymal transition (EMT) molecules, such as E-cadherin, N-cadherin, vimentin, matrix metalloproteinase (MMP)-3 and MMP-9, were determined by Western blot.RESULTS: Chronic hypoxia significantly increased the viability, proliferation, and invasion and migration abilities of MCF-7 cells in vitro, enhanced the anchorage-independent growth, facilitated cellular polarization alteration in Matrigel-3D culture, and promoted cancer metastasis in vivo. Hypoxia up-regulated HIF-1, activated GSK-3β, down-regulated E-cadherin and increased the protein levels of N-cadherin, vimentin, MMP-3 and MMP-9. CONCLUSION: Chronic hypoxia enhances the aggressiveness of breast cancer cells probably through EMT.     

Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

miR-556-3p regulates viability,migration and invasion of endometrial cancer cells by targeting SASH1

AIM To investigate whether microRNA-556-3p （miR-556-3p） regulates the viability, migration and invasion of endometrial cancer cells by targeting SASH1 gene. METHODS The expression of miR-556-3p, and the mRNA and protein levels of SASH1 in endometrial cancer tissues were detected by RT-qPCR and Western blot. Anti-miR-556-3p or pcDNA-SASH1 was transfected into endometrial cancer Ishikawa cells. The cell viability was detected by MTT assay, the migration and invasion abilities of the cells were detected by Transwell chamber method, and the protein expression levels of cyclin D1, p21, matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Western blot. StarBase prediction and dual-luciferase reporter experiments were used to analyze the targeting relationship between miR-556-3p and SASH1. Anti-miR-556-3p and si-SASH1 were co-transfected into the Ishikawa cells, and their effects on cell viability, migration and invasion were examined by the methods described above. RESULTS Compared with adjacent tissues, the expression of miR-556-3p in endometrial cancer tissues was increased significantly, and the expression of SASH1 at mRNA and protein levels was decreased significantly （P<0.05）. Inhibition of miR-556-3p expression or induction of SASH1 over-expression obviously reduced the viability of Ishikawa cells, the number of migratory cells, the number of invasive cells and the protein levels of cyclin D1, MMP-2 and MMP-9, and dramatically increased the protein level of p21 （P<0.05）. miR-556-3p targeted SASH1 and negatively regulated its expression. Knock-down of SASH1 expression reversed the inhibitory effect of miR-556-3p expression inhibition on the viability, migration and invasion of Ishikawa cells. CONCLUSION Inhibition of miR-556-3p expression suppresses the viability, migration and invasion of endometrial cancer cells. The mechanism is related to the regulation of its target gene SASH1.     

PAK6 signaling attenuates migratory and invasive abilities of non-small cell lung cancer cells

AIM：To investigate the roles of p21-activated kinase 6 (PAK6) and its target miRNA on the migratory and invasive abilities of non-small cell lung cancer cells. METHODS：miRNA candidates targeting PAK6 were predicted by a target prediction program. The expression of PAK6 was measured by real-time PCR and Western blotting after A549 cells were transfected with miR-23a mimics or inhibitory oligonucleotides. Luciferase reporter assay was used to determine whether PAK6 was the direct target of miR-23a. The abilities of cell migration and invasion were detected by Matrigel invasion assay and Transwell migration assay. The expression of PAK6 and matrix metalloproteinase 9 (MMP-9) was analyzed by Western blotting after A549 cells were transfected with siPAK6 or miR-23a mimics. RESULTS：miR-23a was identified by a target prediction program. Exogenetic over-expression of miR-23a resulted in a remarkable decrease in PAK6 expression (69%), whereas miR-23a inhibitory oligonucleotides induced pronounced increase in PAK6 expression (52%). The luciferase activity was significantly inhibited by 52% in wild-type PAK6 group, while there was no significant difference in the mutation group. The mRNA level of PAK6 had no change as detected by real-time PCR. Matrigel invasion assay and Transwell migration assay demonstrated there exogenetic over-expression of miR-23a markedly reduced the migration and invasion of PC-3 cells (73% and 59%, respectively). The MMP-9 expression remarkably decreased by 85％ and 76％ in the A549 cells transfected with siPAK6 and miR-23a mimics, respectively. CONCLUSION：miR-23a inhibits the migration and invasion of non-small cell lung cancer cells by repressing PAK6-MMP-9 signaling pathway.     

Role of HMGA2 in epithelial-mesenchymal transition of gastric cancer cells

AIM:To investigate the role of HMGA2 in the epithelial-mesenchymal transition (EMT) in gastric cancer cells. METHODS:The expression of HMGA2 in human gastric cancer cell lines with different degrees of differen-tiation (MKN45, MKN28 and SGC7901) and immortalized human gastric epithelial cell line GES-1 was determined by Western blot and RT-qPCR. pcDNA3.0-HMGA2 plasmid was transfected into the MKN28 cells by liposome method. Transfection of si-HMGA2 interference fragments into MKN45 cells was also performed. The transfection efficiency was evaluated by Western blot and RT-qPCR. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the cell viability were measured by CCK-8 assay. The effects of HMGA2 over-expression in the MKN28 cells on the cell migration and invasion abilities were detected by wound healing and Transwell invasion assays. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the expression of EMT-related markers E-cadherin, N-cadherin, vimentin at mRNA and protein levels were determined by RT-qPCR and Western blot. The changes of Wnt/β-catenin signaling pathway-related molecules in the MKN28 cells with HMGA2 over-expression were also determined by RT-qPCR. RESULTS:The expression levels of HMGA2 were quite different in different differentiation levels of gastric cancer cells (P<0.05). The increased expression level of HMGA2 in MKN28 cells inhibited the cell viability (P<0.05), while the decreased expression level of HMGA2 in MKN45 cells promoted the cell viability (P<0.05). The increased expression level of HMGA2 in MKN28 cells promoted cell migration and invasion (P<0.05), changed the expression of EMT-related markers (P<0.05), while the decreased expression level of HMGA2 in the MKN45 cells changed the expression of EMT-related markers (P<0.05). The increased expression level of HMGA2 in the MKN28 cells significantly increased the mRNA levels of β-catenin in the Wnt/β-catenin pathway and the downstream molecules c-Myc and cyclin D1 (P<0.05). CONCLUSION:HMGA2 is closely related to the migration and invasion abilities of gastric cancer cells. Moreover, it promotes the EMT process of gastric cancer cells by activating Wnt/β-catenin pathway.     

miR-137 regulates invasion,migration and apoptosis of breast cancer MDA-MB-231 cells through TWIST1

AIM: To investigate the effect and its molecular mechanism of microRNA-137(miR-137) on the invasion, migration abilities and apoptosis of breast cancer cells. METHODS: miR-137 mimimics were transfected into the breast cancer MDA-MB-231 cells. The expression of miR-137 was detected by RT-qPCR. Apoptosis was analyzed by flow cytometry. The invasion and migration abilities were detected by Transwell assays. The protein levels of matrix metalloproteinase 9 (MMP-9), cleaved caspase-3 (C-caspase-3) and Bax were determined by Western blot. Bioinformatics software was used to predict that TWIST1 might be the target gene of miR-137 and then it was conformed by luciferase reporter gene identification. The effect of miR-137 mimics on TWIST1 protein expression was evaluated by Western blot. TWIST1 over-expression vector and miR-137 mimics were co-transfected into the MDA-MB-231 cells, and then the apoptosis, invasion, migration abilities and the protein levels of MMP-9, C-caspase-3 and Bax were determined. RESULTS: In the miR-137 mimics transfected MDA-MB-231 cells, the expression level of miR-137 and the apoptosis rate were increased, the cell invasion and migration abilities were decreased, the protein levels of C-caspase-3 and Bax were increased, the protein expression of MMP-9 was decreased (P<0.05). In addition, the target regulation of TWIST1 by miR-137 was identified by luciferase reporter assay. Moreover, the expression of TWIST1 in the MDA-MB-231 cells was inhibited by miR-137 mimics. Compared with the MDA-MB-231 cells co-transfected with negative control vector and miR-137 mimics, the protein expression levels of TWIST1 and MMP-9 in the MDA-MB-231 cells co-transfected with TWIST1 over-expression vector and miR-137 mimics were increased, the protein levels of C-caspase-3 and Bax and the apoptosis rate were decreased, the cell invasion and migration abilities were increased. CONCLUSION: miR-137 inhibits the invasion, migration abilities and induces apoptosis of breast cancer cells through targeting TWIST1.     

Effect of BMP9 over-expression on migration and invasion of human lung squamous-cell carcinoma NCI-H520 cells

AIM: To investigate the effect of bone morphogenetic proteins 9(BMP9) on the migration and invasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism. METHODS: The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial (HBE) cells was detected by RT-PCR and Western blot. The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expression of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot. The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays. The mRNA and protein levels of the migration-related factor matrix metalloproteinase 2(MMP2) were detected by RT-PCR and Western blot. The level of phosphorylated Smad1/5(p-Smad1/5) was detected by Western blot. Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9. The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays. RESULTS: The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells. After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly decreased, and the mRNA and protein levels of MMP2 were decreased. Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability. CONCLUSION: Over-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells. The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.     

Sinomenine inhibits viability,migration and invasion of human ovarian cancer SKOV3 cells

AIM:To investigate the effect of sinomenine on the viability, migration and invasion of human ovarian cancer SKOV3 cells and its possible mechanism. METHODS:The SKOV3 cells were treated with sinomenine at different concentrations for 12 h, 24 h and 48 h. CCK-8 assay was employed to detect the effects of sinomenine on the viability of the SKOV3 cells. Flow cytometry was used to analyze the cell cycle distribution. The cell migration and invasion abilities were measured by Transwell assay. Western blot was used to determine the protein levels of cyclin A, cyclin D1, E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS:Sinomenine remarkably inhibited the viability of SKOV3 cells and IOSE80 cells in a time-dependent and dose-dependent manner (P<0.05), and the IC₅₀ values of 48 h were 2.12 mmol/L and 17.35 mmol/L, respectively. In a dose-dependent manner, sinomenine induced G₀/G₁ and S phase arrest in SKOV3 cells (P<0.05), suppressed the migration and invasion abilities of SKOV3 cells (P<0.05), down-regulated the protein levels of cyclin A, cyclin D1 and MMP-9 (P<0.05), and up-regulated the protein level of E-cadherin (P<0.05). CONCLUSION:Sinomenine inhibits the viability, migration and invasion of human ovarian cancer SKOV3 cells most likely via down-regulation of the protein levels of cyclin A, cyclin D1 and MMP-9, and up-regulation of the protein level of E-cadherin.     

Linarin inhibits migration and invasion abilities of human breast cancer MDA-MB-231 cells through IKK/NF-κB signaling pathway

AIM: To investigate the effect of linarin (LIN) on the migration and invasion abilities of human breast cancer MDA-MB-231 cells and its underlying mechanism. METHODS: MCF-7, MDA-MB-231 and MCF-10A cells were cultured in vitro and treated with LIN at 5, 10, 20, 40, 80 and 160 μmol/L for 24 h, and the cell proliferation was measured by CCK-8 assay and colony formation assay. The protein levels of Snail, E-cadherin, matrix metalloproteinase-9 (MMP-9), IκBα, p-IKKα/β and p-p65 were determined by Western blot. RESULTS: LIN remarkably reduced the viability of MDA-MB-231 cells in a dose-dependent manner (P<0.05), and the IC₅₀ was 55.89 μmol/L for 24 h. LIN decreased the colony formation rate of MDA-MB-231 cells at the concentration of 20 μmol/L (P<0.05). After exposed to LIN at 5 μmol/L and 10 μmol/L for 24 h, the migration and invasion abilities of the MDA-MB-231 cells were significantly reduced (P<0.05), the protein expression levels of E-cadherin and IκBα were up-regulated (P<0.05), the protein expression levels of Snail and MMP-9 were down-regulated (P<0.05), and the phosphorylation levels of IKKα/β and p65 were decreased (P<0.05) in comparison with the control group. Meanwhile, IKK-16 (IKKα/β inhibitor) and PDTC (NF-κB inhibitor) also down-regulated the protein expression levels of Snail and MMP-9 (P<0.05), and up-regulated the protein expression level of E-cadherin (P<0.05). CONCLUSION: LIN down-regulates the protein expression levels of Snail and MMP-9, and up-regulates the protein expression level of E-cadherin most likely through inhibiting IKK/NF-κB signaling pathway, and ultimately lead to decreases in the migration and invasion abilities of MDA-MB-231 cells.