miR-496 over-expression inhibits growth and metastasis in colon cancer cells

AIM: To investigate the effect of miR-496 over-expression on the growth and metastasis of colon cancer cells and its molecular mechanism.METHODS: The proteins interacting with miR-496 were screened by bioinformatic method. The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines, HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM460 were detected by real-time PCR and Western blot. HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control. The cell viability, lactate dehydrogenase (LDH) leakage, and colony formation and metastatic abilities were determined by MTT assay, LDH assay, colony formation assay and Transwell method, respectively. The promoter activity of miR-496 was measured using luciferase reporter gene assay. The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, low-density lipoprotein receptor-related protein 6(LRP6), p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS: Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells. Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected. In contrast, high β-catenin expression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells. Compared with control group, the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells (P<0.05). The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group. miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced. siRNA- or over-expressed miR-496-mediated β-catenin down-regulation inhibited MMP-7 and MMP-9 expression, but promoted TIMP-2 expression.CONCLUSION: The expression level of miR-496 in the colon cancer cells is low, but in the normal colonic epithelial cells is high. miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells.     

Effects of human bone morphogenetic protein 2 and 9 on proliferation,apoptosis and migration of human gastric carcinoma MNK-45 cells

AIM: To investigate the effects of human bone morphogenetic protein 2 (BMP2) and BMP9 on the proliferation, apoptosis and migration of human gastric carcinoma cell line MNK-45. METHODS: Immunocytochemical staining, MTT assay, wound-healing test, Transwells migration test, Hoechst 33258 staining and flow cytometry (FCM) were used to determine the infection of AdBMP2 and AdBMP9 on the proliferation, apoptosis and migration of MNK-45 cells. The expression of GSK-3β (including p-GSK-3β and total GSK-3β) and β-catenin in MNK-45 cells was also detected by Western blotting. RESULTS: The proliferation of MNK-45 cells was inhibited from the third day on and in a time-dependent manner after infected with AdBMP2 and AdBMP9. The results of Hoechst 33258 staining and FCM proved that apoptosis rates in BMP2 group and BMP9 group were higher than that in GFP group. Both wound-healing test and Transwell experiment indicated that up-regulating the expression of BMP2 and BMP9 inhibited the migration of MNK-45 cells. The phosphorylation levels of GSK-3β in BMP2 group and BMP9 group were higher than that in GFP group. However, no significant change of β-catenin among groups was observed. CONCLUSION: Up-regulation of BMP2 and BMP9 expression inhibits the proliferation of MNK-45 cells.     

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.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin 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 GSK-3β knockdown by RNA interference on formation of keloid in vitro

AIM: To study the suppressive effect of glycogen synthase kinase-3β (GSK-3β) knockdown by RNA interference on the formation of keloid. METHODS: Human keloid fibroblasts (KFB) in vitro were transfected with 3 pairs of specific GSK-3β small interfering RNA (siRNA). The best siRNA to inhibit the GSK-3β expression in human KFB was screen by RT-PCR and Western blot. The expression of GSK-3β and related proteins at mRNA and protein levels in the KFB was determined by RT-PCR and Western blot.RESULTS: The GSK-3β siRNA1434 remarkably inhibited the expression of GSK-3β at mRNA and proteins levels in the human KFB. After transfection with GSK-3β siRNA, the protein levels of β-catenin, p-GSK-3β, Wnt2 and cyclin D1 were all decreased. KFB growth became slow. With the extension of time, the inhibition of cell growth increased, and the cell doubling time was significantly delayed. CONCLUSION: siRNA targeting GSK-3β efficiently knocks down the expression of GSK-3β in the human KFB, and inhibits the activation of Wnt signaling pathway, thus inhibiting the growth of keloid. GSK-3β may be a potential therapeutic target for keloid.     

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.     

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.     

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.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 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.     

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.     

Effects of sulindac on oxidative stress in an autistic model induced by prenatal exposure to valproic acid

AIM: To investigate the effects of sulindac on oxidative stress in autism. METHODS: With an autistic model induced by prenatal exposure to valproic acid (VPA), we detected the expression of the signaling molecules of canonical Wnt pathway in the prefrontal cortex (PFC) and hippocampus (HC) of autistic rats treated with sulindac. The protein expression levels of glycogen synthase kinase 3β (GSK-3β), β-catenin and 4-hydroxynonenal (4-HNE) were observed by Western blotting. The mRNA expression of thioredoxin(Trx)1 and Trx2 was assessed by semi-quantitative RT-PCR.RESULTS: The protein level of GSK-3β and mRNA levels of Trx1 and Trx2 were lower, whereas the protein expression levels of β-catenin and 4-HNE were higher in VPA group than those in control group. In contrast, the protein levels of GSK-3β were significantly higher in the animals treated with both VPA and sulindac than those in VPA group, while the levels of β-catenin and 4-HNE were decreased.CONCLUSION: Sulindac attenuates oxidative stress in the pathogenesis of autism, suggesting the up-regulation of the Wnt/β-catenin signaling pathway disrupts oxidative homeostasis and further facilitates susceptibility to autism.     

Effects of ATP-competitive GSK-3 inhibitor on multidrug resistance of human esophageal squamous-cell carcinoma cells

AIM: To study the changes and correlations of β-catenin, multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp), Bcl-2 and the free ATP level in esophageal squamous-cell carcinoma (ESCC) cell line EC-109 induced by ATP-competitive glycogen synthase kinase(GSK-3) inhibitor 6-bromoindirubin-3'-oxime (BIO). METHODS: The methods of flow cytometry, immunofluorescence, cytochemistry and ATP assay were used to study the expression levels of MRP2, P-gp, β-catenin and Bcl-2, the efflux capability of ATP-binding cassette (ABC) transporters, and the free ATP level in ESCC cells. RESULTS: After induced by BIO, up-regulation of β-catenin and Bcl-2 expression in cytoplasm and accumulation of β-catenin in nucleus were found in ESCC cells. The expression of MRP2 was up-regulated in cytoplasm and cell membrane. On the contrary, the expression of P-gp was down-regulated in cytoplasm and cell membrane. The free ATP level and the efflux capability of ABC transporters increased in ESCC cells. CONCLUSION: The multidrug resistance of ESCC cells in enhanced by BIO treatment.     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

Role of β-catenin in apoptosis of pancreatic acinar cells induced by cae-rulein

AIM: To study the role of β-catenin in the apoptosis of pancreatic acinar cells induced by cae-rulein. METHODS: Rat pancreatic acinar AR42J cells were treated with caerulein. The expression of β-catenin at mRNA and protein levels in the AR42J cells was determined by real-time PCR and Western blot. The β-catenin over-expression vector was transfected into AR42J cells. After treatment with caerulein, the over-expression effect was evaluated by real-time PCR and Western blot. The changes of cell viability were measured by MTT assay. The leakage rates of lactate dehydrogenase (LDH) and amylase (AMY) were measured by binitrophenyl hydrazine method and iodine starch colorimetry, respectively. The apoptosis was analyzed by flow cytometry. The protein levels of endoplasmic reticulum stress protein CHOP and cleaved caspase-12 in the AR42J cells were determined by Western blot. RESULTS: The expression of β-catenin at mRNA and protein levels in the AR42J cells was decreased after treatment with caerulein (P<0.05). The expression of β-catenin in the AR42J cells was significantly increased by transfection with β-catenin over-expression vector. The viability of AR42J cells after treatment with caerulein was reduced, while the leakage rates of LDH and AMY, the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the cells were increased (P<0.05). Over-expression of β-catenin enhanced the viability of AR42J cells after treatment with caerulein, reduced the leakage rates of LDH and AMY, and decreased the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the AR42J cells. CONCLUSION: β-Catenin significantly inhibits the apoptosis of pancreatic acinar cells induced by caerulein. The mechanism is related to the reduction of endoplasmic reticulum stress.     

Effects of stachydrine hydrochloride on experimental acute cerebral infarction in rats

AIM: To observe the therapeutic effect of stachydrine hydrochloride on experimental acute cerebral infarction in rats and to explore the underlying mechanisms. METHODS: SD rats (n=75) were randomly divided into 5 groups: sham group, cerebral infarction model group, and stachydrine hydrochloride (10 mg/kg, 20 mg/kg and 40 mg/kg) treatment groups. After the establishment of cerebral infarction model, the rats were given stachydrine hydrochloride at dose of 10 mg/kg, 20 mg/kg or 40 mg/kg by gavage daily for 14 d. The impairment of neurological function in each group was scored. The cerebral infarction volume and brain water content were measured. Moreover, the protein levels of β-catenin, cyclin D1, glycogen synthase kinase 3β (GSK-3β) and p-GSK-3β in the brain tissues were detected by Western blot. RESULTS: Compared with cerebral infarction group, the score of neurological function impairment, cerebral infarction volume and brain water content were significantly decreased in stachydrine hydrochloride treatment groups. In addition, the protein levels of β-catenin, cyclin D1 and p-GSK-3β were markedly increased after stachydrine hydrochloride treatment. CONCLUSION: Stachydrine hydrochloride protects against experimental acute cerebral infarction through activation of Wnt/β-catenin signaling pathway.     

Cholestane-3β, 5α, 6β-triol induces apoptosis of malignant glioma cells

AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.     

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.     

Effect of wogonin on OSCC cell growth and invasion

AIM: To investigate the molecular mechanism of wogonin on the growth and invasion of oral squamous-cell carcinoma (OSCC) cell line SCC-4. METHODS: After treatment with various doses (0, 20, 40, 60, 80 and 100 mg/L) of wogonin for the indicated time, MTT assay was used to evaluate cell viability. The cell apoptosis was detected by flow cytometry with Annexin V and propidium iodide (Annexin V/PI) double staining. The cell invasion ability was analyzed by Transwell assay. The activation of Wnt/β-catenin signaling molecules was assessed by Western blot. RESULTS: Wogonin inhibited the viability and invasion of SCC-4 cells but promoted cell apoptosis in a dose-and time-dependent manner. Wogonin treatment obviously decreased the activation of β-catenin. Moreover, the expression of downstream targets cyclin D1, matrix metalloproteinase-2 (MMP-2) and MMP-9 were obviously down-regulated, accompanied by the up-regulation of anti-apoptotic protein Bcl-2. Wnt/β-catenin activator LiCl remarkably attenuated the inhibitory effect of wogonin on the activation of Wnt/β-catenin signaling molecules. Importantly, the inhibition of cell growth and invasion ability by wogonin treatment was dramatically attenuated after LiCl exposure. CONCLUSION: Wogonin blocks SCC-4 cell growth and invasion mainly by regulating Wnt/β-catenin signaling, indicating that it is a potential suppressor for OSCC and may be a potential target for the development of anti-OSCC therapy.