Promoting cell viability and migration of gastric cancer cells by PRRX2 via activation of Wnt/β-catenin signaling pathway

AIM: To study the effect of paired-related homeobox 2 (PRRX2) gene on the viability and migration ability of gastric cancer cells, and to analyze the underlying mechanism of regulating Wnt/β-catenin signaling pathway.METHODS: The expression of PRRX2 in gastric cancer and normal gastric tissue and the correlation between PRRX2 expression in gastric cancer tissues with the overall survival rate of gastric cancer patients were analyzed by bioinformatics. The small interfering RNA (siRNA) and over-expressed plasmids of PRRX2 were transfected into gastric cancer cells MGC-803 and SGC-7901, respectively. MTT assay and Transwell assay were used to detect the viability and migration ability of gastric cancer cells. Western blot and TOPflash/FOPflash dual-luciferase reporter gene assay were used to detect the activity of Wnt/β-catenin signaling pathway. Co-immunoprecipitation was used to detected the interaction between PRRX2 and β-catenin proteins.RESULTS: Knockdown of PRRX2 attenuated the viability and migration ability of gastric cancer cell line MGC-803 (P<0.05). Over-expression of PRRX2 enhanced the viability and migration ability of SGC-7901 cells (P<0.05), increased the protein levels of β-catenin, c-Myc and cyclin D1 (P<0.05) and the activity of TOPflash/FOPflash dual-luciferase reporter gene (P<0.05). PRRX2 interacted with β-catenin protein in gastric cancer cells.CONCLUSION: PRRX2 promotes the viability and migration ability of gastric cancer cells, which may be related to Wnt/β-catenin signaling pathway.     

Juglone inhibits epithelial-mesenchymal transition of prostate cancer cells by regulating Wnt/β-catenin/Snail signaling pathway

AIM: To investigate the mechanism of juglone on epithelial-mesenchymal transition in prostate cancer cells. METHODS: Human prostate cancer LNCaP cells were divided into control group (without juglone), 12.5 μmol/L juglone group and 25 μmol/L juglone group. LNCaP cells in the latter 2 groups were treated with juglone for 24 h. The invasion ability of the LNCaP cells was detected by Transwell assay. The protein expression of E-cadherin, vimentin, Snail and β-catenin was determined by Western blot. The LNCaP cells were treated with LiCl and juglone in combination for 24 h, and the protein expression of Snail and E-cadherin was detected by Western blot.RESULTS: The results of Trans-well invasion assay showed that the invasion ability in juglone groups was significantly decreased (P<0.01). The protein expression of E-cadherin in the LNCaP cells treated with juglone was increased, and the expression levels of vimentin and β-catenin were reduced (P<0.01). Treatment with LiCl significantly attenuated the inhibitory effect of juglone on Snail expression and subsequent down-regulation of E-cadherin expression. CONCLUSION: Juglone inhibits the epithelial-mesenchymal transition by inhibiting the Wnt/β-catenin/Snail signaling pathway in the LNCaP cells.     

Over-expression of SCUBE2 suppresses epithelial-mesenchymal transition through Wnt/β-catenin signaling pathway in colorectal cancer cells

AIM: To study the effect of SCUBE2 on epithelial-mesenchymal transition (EMT) in colorectal cancer cells and its mechanism. METHODS: The expression of SCUBE2 in human colorectal cancer cell line HCT116 and normal colonic cell line FHC was detected by real-time PCR and Western blot. HCT116 cells were transfected with GV144-SCUBE2 to over-express SCUBE2, and then the cell viability, migration, and apoptosis were determined by MTT assay, Transwell assay and flow cytometry, respectively. The expression of EMT markers (E-cadherin, vimentin, and Snail), β-catenin, c-Myc and cyclin D1 in the HCT116 cells was analyzed by real-time PCR or Western blot after transfection with GV144-SCUBE2 for 6 h, followed by the stimulation of 10 μg/L recombinant TGF-β1 protein for 48 h. Additionally, the EMT process of HCT116 cells, which were stimulated by TGF-β1, over-expressed SCUBE2, and treated with Wnt/β-catenin pathway activator lithium chloride (LiCl) or inhibitor XAV93920, was analyzed by Western blot. RESULTS: Compared with FHC cells, the expression of SCUBE2 in the HCT116 cells was significantly decreased. The viability and migration ability of the HCT116 cells were suppressed by SCUBE2 over-expression, but the apoptosis was not markedly changed. Elevated expression of SCUBE2 increased E-cadherin expression, and decreased the expression of vimentin, Snail, β-catenin, c-Myc and cyclin D1 induced by TGF-β1. Treatment with LiCl blocked but treatment with XAV93920 enhanced the effects of SCUBE2 on EMT. CONCLUSION: Over-expression of SCUBE2 may inhibit the cell growth and migration, and suppress EMT through Wnt/β-catenin signaling pathway.     

Expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and its clinical significance

AIM: To evaluate the expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and the significance. METHODS: The patients with breast cancer (n=150) in our hospital from January 2015 to January 2017 were selected as study object. The tumor tissue samples of these patients were obtained from paraffin section of breast cancer by surgical resection with complete clinicopathological data. The corresponding paracancerous tissue sam-ples were taken from the non-tumor tissue samples from the above breast cancer patients, which were 0.5~1 cm away from the tumor tissue. The methods of real-time PCR and Western blot were performed to examine the expression of Wnt-1 and β-catenin at mRNA and protein levels. Human breat cancer MCF-7 cells were divided into 3 groups:control group (MCF-7 cells without treatment), agonist group[MCF-7 cells+Wnt3a (1 mg/L)] and antagonit group[MCF-7 cells+DKK1 (16 μmol/L)]. The expression of Wnt-1 and β-catenin at mRNA and protein levels was detected by real-time PCR and Western blot. RESULTS: Compared with the paracancerous tissues, the expression levels of Wnt-1 and β-catenin were higher in tumor tissues at mRNA and proteins levels (P<0.05). Notably, the positive expression rates of Wnt-1 and β-catenin were significantly higher in tumor tissues than that in the paracancerous tissues. Furthermore, Wnt-1 expression was associated with tumor metastasis (χ²=5.352, P=0.021), tumor stage (χ²=9.412, P=0.002) and tumor size (χ²=9.412, P=0.002). In addition, β-catenin expression was also associated with tumor metastasis (χ²=9.851, P=0.002) and tumor stage (χ²=5.661, P=0.017). Compared with control group, the expression of Wnt-1 and β-catenin at mRNA and protein levels in agonist group was increased (P<0.05),while that in antagonist group was decreased (P<0.05). CONCLUSION: The expression levels of Wnt-1 and β-catenin related with Wnt/β-catenin signaling pathway are increased in the breast cancer, which are closely related to the malignant state of the tumor.     

Effect of EZH2 regulating Wnt/β-catenin signaling pathway on apoptosis of brain glioma cells

AIM: To investigate the effect of enhancer of zeste homolog 2 (EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87, H4 and U251 and normal human astrocytes (NHA) was detected by RT-qPCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway, the apoptosis rate was measured by flow cytometry, and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87, H4 and U251 were significantly higher than those in NHA (P<0.05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells (P<0.05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells, the apoptotic rate was significantly increased, and the activity of caspase-3 was significantly increased in the cells (P<0.05). Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2, and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Crosstalk between Wnt/β-catenin and NF-κB signaling #br# pathways during wound healing

Both Wnt/β-catenin and NF-κB signaling pathways are conserved pathways that regulate a variety of biological processes throughout the development and adult lifetime of mammals. Recent findings suggest that the 2 signaling pathways can cross-regulate each other. Several studies show that there are both positive and negative regulations between the 2 pathways. Some major components are involved in the cross-regulation of the 2 pathways such as Wnt proteins, DKK1, E-cadherin, GSK-3β, IKKs/IκB and so on. By reviewing the literature, we conjecture that the crosstalk between the 2 pathways regulate the wound healing process by intervening in the inflammation, fibroblast and angiogenesis, but the concrete mechanism is still unknown.     

Expression changes of Wnt/β-catenin signaling pathway in diabetic ulcer

AIM: To observe the effect of Wnt/β-catenin signaling pathway on diabetic ulcer. METHODS: Diabetic animal model was established in the female Wistar rats by intraperitoneal injection of low-dose streptozotocin following high-fat diet feeding. A circular wound was made on the dorsum of the rats in both control group and diabetic group. The condition of wound healing was recorded and the structures of the wound tissues were observed by HE staining in the 2 groups at 3, 7 and 14 d after wounding. The expression of β-catenin, GSK-3β and Rspo-3 at mRNA and protein levels in the wound tissues was detected by RT-PCR and ELISA. RESULTS: In diabetic group, the wound healing rate was lower (P<0.05), and the inflammatory cells, fibroblast cells and new capillaries in the wound tissues were fewer than those in control group. The expression of β-catenin and Rspo-3 at mRNA and protein levels in the wound tissues in control group was significantly higher than those in diabetic group, and the expression of GSK-3β was exactly the opposite (P<0.05). CONCLUSION: The down-regulation of Wnt/β-catenin probably resultes from the decreased level of Rspo-3, which may be one of the reasons for delaying the diabetic ulcer healing.     

Effects of Wnt/β-catenin signal pathway on asthma airway remodeling

AIM: To explore the effect of Wnt/β-catenin signaling pathway in airway smooth muscle cells (ASMC) on asthmatic airway remodeling.METHODS: The asthmatic airway remodeling model in rats was established and the ASMC was isolated and cultured. The protein expression of β-catenin, glycogen synthase kinase-3β (GSK-3β), c-Myc and cyclin D1 in the ASMC was determined by Western blot. After depressing the interaction between β-catenin and p300/CBP, the cell activity was measured by CCK-8 assay and the change of cell cycle distribution was analyzed by flow cytometry. Meanwhile, the protein expression of c-Myc and cyclin D1 in the ASMC was determined by Western blot after inhibiting P38 mitogen-activated protein kinase (MAPK) activity.RESULTS: The protein levels of β-catenin, c-Myc and cyclin D1 were significantly increased in asthma group while the protein level of GSK-3β was decreased in the same group (P<0.05). After depressing the interaction between β-catenin and p300/CBP, the cell activity of ASMC was decreased in asthma group compared with control group (P<0.05), and the change of the cell cycle distribution in asthma group was also more obvious (P<0.05). After inhibiting P38 MAPK activity, the protein levels of c-Myc and cyclin D1 were all decreased compared with control group in ASMC asthma and control rats (P<0.05).CONCLUSION: Wnt/β-catenin signaling pathway may participates in airway remodeling in asthma by increasing the protein expression of c-Myc and cyclin D1, reacting with the P38 MAPK signaling pathway and regulating the growth of ASMC.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

β-estradiol promotes invasion and migration of lung cancer A549 cells via ERβ-mediated ERK1/2 membrane-initiated steroid signaling

AIM: To investigate the regulatory mechanism of β-estradiol in the invasion and migration of lung cancer A549 cells. METHODS: Breast cancer MCF-7 cells and lung cancer A549 cells were cultured in vitro. The MCF-7 cells were used as the estrogen receptor (ER) positive expression cell model. Real-time PCR and immunofluorescence were employed to measure the expression level and the localization of ER in A549 cells. The phosphorylation of ERK1/2 upon β-estradiol stimulation was quantified by Western blot. The invasion and migration abilities of A549 cells upon β-estradiol stimulation with or without ERK1/2 inhibitor PD98059 were measured by Transwell and Cell-IQ assays. RESULTS: ERβ was the dominant ER subtype in the A549 cells and primarily comprised of ERβ2 and ERβ5. Immunofluorescence revealed that ERβ expression was mainly localized in the cytoplasm. β-estradiol induced phosphorylation of ERK1/2 and promoted the invasion and migration of the cells. Inhibition of ERK1/2 signaling reversed β-estradiol-promoted invasion and migration of A549 cells. CONCLUSION: ERβ-mediated membrane-initiated steroid signaling is involved in the process of β-estradiol-promoted invasion and migration of A549 cells, through which ERK1/2 signaling plays a pivotal role.     

Dickkopf-1 silencing inhibits invasion and epithelial-mesenchymal transition in gastric carcinoma cells by down-regulating β-catenin

AIM: To explore the expression of Dickkopf-1 (DKK1) in human gastric carcinoma cells, and the influences of DKK1 gene silencing on cell invasion. METHODS: The levels of DKK1 in the human gastric mucosa cell line GES-1 and gastric carcinoma cell lines MKN-45 and SGC-7901 were detected by real-time PCR and Western blot. DKK1 gene was silenced by RNA interference, which was verified by real-time PCR, Western blot and ELISA. The cell invasion ability was determined by Transwell assay, and the cell proliferation was inhibited by mitomycin C. The levels of E-cadherin, N-cadherin, vimentin and β-catenin were determined by real-time PCR and Western blot. RESULTS: The expression of DKK1 was significantly higher in MKN-45 cells and SGC-7901 cells than that in GES-1 cells, indicating that DKK1 expression was obviously increased in gastric carcinoma cells. After successful silencing of DKK1 gene in the MKN-45 cells and SGC-7901 cells, the cell invasion ability was markedly decreased in a time-dependent pattern with increased expression of E-cadherin and decreased expression of N-cadherin and vimentin, indicating that DKK1 silencing dramatically inhibited gastric carcinoma cell invasion and epithelial-mesenchymal transition (EMT). The introduction of exogenous recombinant DKK1 (rDKK1) demonstrated the promoting effect of DKK1 on gastric carcinoma cell invasion and EMT. In addition, the inhibitory effects of DKK1 silencing on gastric carcinoma cell invasion and EMT were fulfilled by down-regulating β-catenin. CONCLUSION: The expression of DKK1 is significantly increased in human gastric carcinoma cells. Silencing of DKK1 markedly inhibits gastric carcinoma cell invasion and EMT by down-regulating β-catenin.     

Ginsenoside Rg1 promotes growth and extracellular matrix synthesis in degenerative human lumbar nucleus pulposus cells by inhibiting Wnt/β-catenin pathway

AIM: To explore the role of ginsenoside Rg1 in the growth of degenerative human lumbar nucleus pulposus cells (HNPCs). METHODS: Cultured HNPCs were subjected to oxygen-glucose deprivation (OGD) to mimic the micro-environment of degenerative HNPCs. The morphological changes of the cells in control group and OGD group were observed under optical microscope. The cells were treated with ginsenoside Rg1 at concentrations of 25, 50 and 100 μmol/L. The expression of collagen Ⅱ and aggrecan at mRNA and protein levels was determined by real-time PCR and Western blot analysis. The cell viability was measured by CCK-8 assay. The mRNA level of Ki67 was detected by real-time PCR. The apoptosis was analyzed by flow cytometry. The activity of caspase-3 was measured by a caspase-3 kit. The expression of Wnt/β-catenin pathway-related proteins was determined by Western blot. Furthermore, the expression of Wnt/β-catenin pathway-related proteins, the cell viability and apoptosis, and the expression of extracellular matrix synthesis proteins were assessed after the cells were co-treated with LiCl and 100 μmol/L ginsenoside Rg1. RESULTS: Normal HNPCs attached on the cell culture plate faster, and were almost round with rich cytoplasm. However, the cell adherence was slower, and the cells were long fusiform with decreased cytoplasm after OGD treatment, indicating that the model of degenerative HNPCs was successfully established. Compared with normal HNPCs, the expression of collagen Ⅱ and aggrecan at mRNA and protein levels was decreased in OGD group (P<0.05), which was then increased after the cells were treated with ginsenoside Rg1 at 25, 50 and 100 μmol/L (P<0.05). Compared with normal HNPCs, the cell viability and Ki67 expression were decreased in OGD group (P<0.05), which were increased after treatment with ginsenoside Rg1 (P<0.05). Meanwhile, the apoptotic rate and caspase-3 activity were significantly increased in OGD-treated cells (P<0.05), which were decreased after treatment with ginsenoside Rg1 (P<0.05). In addition, the activation of Wnt/β-catenin pathway was also inhibited by ginsenoside Rg1 treatment at dose of 100 μmol/L (P<0.05). LiCl, a Wnt/β-catenin pathway agonist, obviously decreased the protective effects of ginenoside Rg1 on OGD-induced cells (P<0.05), indicating that the Wnt/β-catenin pathway was involved in the protective effects of ginenoside Rg1 on degenerative HNPCs. CONCLUSION: Ginsenoside Rg1 promotes growth and extracellular matrix synthesis of degenerative HNPCs through inhibiting Wnt/β-catenin pathway. This study will provide a new idea for prevention and treatment of degenerative HNPCs.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

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.     

Effects of RARG on viability and migration ability of gastric cancer cells

AIM:To investigate the effect of retinoic acid receptor gamma (RARG) on the viability and migration ability of gastric cancer cells. METHODS:The expression of RARG in gastric cancer and normal gastric tissues and its correlation with the overall survival rate of gastric cancer patients were analyzed by bioinformatics. The expression of RARG was promoted and inhibited by over-expression plasmid transfection and RNA interference technique in gastric can-cer cells in vitro, respectively. MTT and Transwell assays were used to detect the effect of RARG on the viability and migration ability of gastric cancer cells. The effect of RARG on regulating the Wnt/β-catenin signaling pathway was evaluated by Western blot and TOP/FOP dual-luciferase reporter assay. The protein interaction of RARG and β-catenin was determined by co-immunoprecipitation and immunofluorescence co-localization assay. RESULTS:Over-expression of RARG enhanced the viability and migration ability of gastric cancer SGC7901 cells (P<0.05). Knockdown of RARG attenuated the viability and migration ability of gastric cancer MGC-803 cells (P<0.05). At the same time, RARG over-expression increased the protein expression levels of β-catenin, c-Myc, cyclin D1, Twist and Snail (P<0.05), and the activity of TOP/FOP dual-luciferase reporter gene (P<0.05). In addition, RARG interacted with β-catenin protein in the gastric cancer cells. CONCLUSION:RARG promotes the viability and migration ability of gastric cancer cells via activating the Wnt/β-catenin signaling pathway, thus playing an important role in the development of gastric cancer.     

Cordycepin inhibits proliferation and migration of gallbladder cancer cell SNU-308 by ERK1/2, Ezrin and Akt signaling pathways

AIM: To investigate the effects of cordycepin on the proliferation and migration abilities of gallbladder cancer cell line SNU-308 and its molecular mechanism. METHODS: The viability of SNU-308 cells treated with cordycepin at different concentrations was measured by MTT assay and the colony formation ability was also detected. The effect of cordycepin on apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of apoptosis and autophagy markers, and the phosphorylation level of Akt, ERK1/2 and Ezrin were evaluated by Western blot. Immunofluorescence staining was also used to analyze the expression level of LC3 after cordycepin treatment. Wound healing assay and Transwell assay were performed to evaluate the migration ability of the SNU-308 cells after cordycepin treatment. Wound healing assay was also used to evaluate the effects of Akt inhibitor, ERK1/2 inhibitor and Ezrin knockdown on the changes of migration ability. RESULTS: Cordycepin significantly inhibited the viability and the ability of colony formation of gallbladder cancer cells (P<0.05). Induction of apoptosis by cordycepin were revealed by flow cytometry (P<0.05). The protein expression of Bcl-2 was down-regulated, while the protein levels of Bax, cytochrome C (Cyto C), Fas, FasL and cleaved caspase-3 were increased and the autophagy marker beclin 1 and the ratio of LC3-Ⅱ/I were upregulated by Western blot analysis (P<0.05). LC3 accumulation in the cytoplasm after cordycepin treatment was demonstrated by immunofluorescence staining. Cordycepin treatment resulted in the inhibition of cell migration were detected by Transwell assay and wound healing assay (P<0.05). The protein levels of p-Akt, p-ERK1/2 and p-Ezrin were down-regulated after cordycepin treatment (P<0.05). Besides, Ezrin knockdown, Akti-1/2 and GDC-0994 all resulted in the inhibition of migration ability (P<0.05). CONCLUSION: Cordycepin induces apoptosis and autophagy to inhibit gallbladder can-cer cell proliferation and migration by regulating ERK1/2, Ezrin and Akt signaling pathways.     

Wnt signaling pathway is involved in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells

AIM：To investigate the changes of Wnt signaling pathway in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：The BMSCs were isolated from SD rats, purified by differential time adherent method and divided into control group and catalpol (1.0 mg/L) group. Flow cytometry was used to detect the proliferation index of BMSCs. The mRNA levels of Wnt3a, Wnt5a, Wnt11 and β-catenin was evaluated by real-time PCR. In addition, the protein expression level of β-catenin was determined by Western blotting. RESULTS：Prolife-ration index was increased from 8.90%±0.46% to 17.93%±1.68% after treatment with catalpol (P<0.01). Compared with control group, the mRNA expression of Wnt5a, Wnt11 and β-catenin was all increased with catalpol treatment. No difference of Wnt3a mRNA expression between control group and catalpol group was observed. Meanwhile, the protein expression of β-catenin was increased in catalpol group compared with control group. CONCLUSION：Catalpol promotes BMSCs going into the cell cycle. Classical and non-classical Wnt signaling pathways are activated in this process.     

Sphingosine kinase 1 enhances invasion and migration of non-small-cell lung cancer cells by ERK1/2 signaling pathway

AIM To explore the effects of sphingosine kinase 1 (SphK1) on the migration and invasion of non-small-cell lung cancer (NSCLC) cells and its mechanism. METHODS Thirty-one tumor specimens, which were surgically resected and routinely histologically confirmed as NSCLC, and matched adjacent lung tissues were selected. Immunohistochemical staining and RT-qPCR were used to detect the expression of SphK1. The pcDNA3.1-SphK1 vector (SphK1 group), empty pcDNA3.1 vector control (NC group), SphK1 siRNA (siSphK1 group) or control siRNA (siNC group) was transfected into human lung adenocarcinoma A549 cells, and the protein levels of SphK1, E-cadherin, fibronectin and p-ERK1/2 were determined by Western blot. The effects of over-expression of SphK1 and inhibition of ERK1/2 on migration and invasion of A549 cells were evaluated by Transwell assays. RESULTS SphK1 was highly expressed in the NSCLC tissues and was associated with tumor stage. SphK1 over-expression significantly promoted the migration and invasion of A549 cells, increased the protein levels of p-ERK1/2 and fibronectin, and decreased the protein expression of E-cadherin (P<0.05), but the opposite result was observed after SphK1 interference. The ERK1/2 inhibitor U0126 significantly inhibited the up-regulation of p-ERK1/2 and fibronectin levels and the down-regulation of E-cadherin expression induced by SphK1 over-expression, and also inhibited the invasion and migration of A549 cells promoted by SphK1 over-expression (P<0.05). CONCLUSION SphK1 may reduce E-cadherin protein levels, increase fibronectin protein levels, and promote the invasion and migration of NSCLC cells through ERK1/2 signaling 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.