Loss of monopolar spindle-binding protein 3B expression promotes colorectal cancer invasiveness by activation of target of rapamycin kinase/autophagy signaling.

BACKGROUND: Monopolar spindle-binding protein 3B (MOB3B) functions as a signal transducer and altered MOB3B expression is associated with the development of human cancers. AIM: To investigate the role of MOB3B in colorectal cancer (CRC). METHODS: This study collected 102 CRC tissue samples for immunohistochemical detection of MOB3B expression for association with CRC prognosis. After overexpression and knockdown of MOB3B expression were induced in CRC cell lines, changes in cell viability, migration, invasion, and gene expression were assayed. Tumor cell autophagy was detected using transmission electron microscopy, while nude mouse xenograft experiments were performed to confirm the in-vitro results. RESULTS: MOB3B expression was reduced in CRC vs normal tissues and loss of MOB3B expression was associated with poor CRC prognosis. Overexpression of MOB3B protein in vitro attenuated the cell viability as well as the migration and invasion capacities of CRC cells, whereas knockdown of MOB3B expression had the opposite effects in CRC cells. At the molecular level, microtubule-associated protein light chain 3 II/I expression was elevated, whereas the expression of matrix metalloproteinase (MMP)2, MMP9, sequestosome 1, and phosphorylated mechanistic target of rapamycin kinase (mTOR) was downregulated in MOB3B-overexpressing RKO cells. In contrast, the opposite results were observed in tumor cells with MOB3B knockdown. The nude mouse data confirmed these in-vitro findings, i.e., MOB3B expression suppressed CRC cell xenograft growth, whereas knockdown of MOB3B expression promoted the growth of CRC cell xenografts. CONCLUSION: Loss of MOB3B expression promotes CRC development and malignant behaviors, suggesting a potential tumor suppressive role of MOB3B in CRC by inhibition of mTOR/autophagy signaling.

SphK1-driven autophagy potentiates focal adhesion paxillin-mediated metastasis in colorectal cancer.

Invasion and metastasis are the main causes of colorectal cancer (CRC)-related death. Accumulating evidence suggested that sphingosine kinase 1 (SphK1) promoted the metastasis of CRC and autophagy played an important role in SphK1 promoting the metastasis of malignancy. However, the mechanism by which SphK1-driven autophagy promotes invasion and metastasis in CRC remains to be clarified. In the present study, immunohistochemical detection showed the expression of SphK1 and paxillin was higher in human CRC tissues than those of normal colorectal mucosal tissues, they were both associated with TNM staging, lymphatic, and distance metastasis. In addition, study of in situ tumor transplantation model in nude mice showed that the suppression of SphK1 inhibited the growth of colonic orthotopic implantation tumors and the expression of paxillin, p-paxillin, LC3 in the tumor. So, SphK1 may promote CRC metastasis via inducing the expression of paxillin expression and its phosphorylation, in vivo. Furthermore, results of CCK8 assay, transwell and wound healing assays showed that SphK1 promoted the viability, invasion, and metastasis of CRC cells. Transmission electron microscopy detection showed that SphK1 is the key factor in autophagy induction in CRC cells. Moreover, western blot examination indicated that the expression of LC3Ⅱ/Ⅰ, paxillin, p-paxillin, MMP-2, and vimentin was enhanced in SphK1-overexpressed CRC cells and suppressed in SphK1 knockdown CRC cells, meanwhile, the expression of E-cadherin was suppressed in SphK1-overexpressed CRC cells and enhanced in SphK1 knockdown CRC cells. Suppression of autophagy by 3MA reversed the expression of paxillin and its phosphorylation in SphK1-overexpressed CRC cells, indicated that SphK1-driven autophagy induced the expression of paxillin and its phosphorylation in CRC cells. Together, these findings reveal that SphK1-driven autophagy may promote the invasion and metastasis of CRC via promoting the expression of focal adhesion paxillin and its phosphorylation.

Ghrelin inhibits IKKß/NF-κB activation and reduces pro-inflammatory cytokine production in pancreatic acinar AR42J cells treated with cerulein.

BACKGROUND: Previous studies have provided conflicting results regarding whether the serum ghrelin concentration can reflect the severity of acute pancreatitis (AP). The present study examined the correlation between the serum ghrelin concentration and AP severity in animal models and investigated whether altered ghrelin expression in pancreatic acinar cells influences IKKß/NF-κB signaling and pro-inflammatory cytokine production. METHODS: Mild or severe AP was induced in rats by intraperitoneal injection of cerulein or retrograde cholangiopancreatic duct injection of sodium taurocholate, respectively. After successful model induction, serum ghrelin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were determined by enzyme-linked immunosorbent assay, and IKKß/NF-κB activation was assessed by immunohistochemistry. Subsequently, stable overexpression or knockdown of ghrelin in AR42J cells was achieved by lentiviral transfection. After transfected cells and control cells were treated with cerulein for 24 h, the TNF-α and IL-1ß levels in the supernatants were determined by enzyme-linked immunosorbent assay, and the expression levels of p-p65, IKKß, and p-IKKß were detected by Western blotting. RESULTS: In rat AP models, AP severity was correlated with increased IKKß/NF-κB activation, pro-inflammatory cytokine production, and ghrelin secretion. The levels of pro-inflammatory cytokines TNF-α and IL-1ß as well as IKKß/NF-κB signaling activity were increased upon knockdown of ghrelin in the AP acinar cell model and decreased with ghrelin overexpression. CONCLUSIONS: Serum ghrelin is related to the severity of AP. Ghrelin may play a protective role in the pathogenesis of AP by inhibiting the pro-inflammatory cytokines and the activation of the IKKß/NF-κB signaling pathway.

Association of tricellulin expression with poor colorectal cancer prognosis and metastasis.

Tricellulin is a tight­junction transmembrane protein that regulates cell­cell interactions. Altered tricellulin expression could promote tumor cell invasions and metastasis in human cancers. The present study assessed tricellulin expression in colorectal cancer tissues for any association with clinicopathological features of colorectal cancer patients and then investigated the underlying molecular events using quantitative proteomic analysis and in vitro experiments. Tissue samples from 98 colorectal cancer patients and 15 volunteers were collected for immunohistochemistry. Colorectal cell lines were used to overexpress or knockdown tricellulin expression in various assays. The data revealed that upregulated tricellulin expression was associated with lymph node and distant metastases and poor prognosis, while tricellulin overexpression promoted colorectal cancer cell migration and invasion in vitro. In contrast, tricellulin knockdown had positive effects on the tumor cells. Furthermore, TMT­LC­MS/MS and bioinformatics analyses revealed that tricellulin was involved in EMT and reduction of apoptosis through the NF­κB signaling pathway. These findings highlight for the first time the significance of tricellulin in colorectal cancer development and progression. Further study may validate tricellulin as a novel biomarker and target for colorectal cancer.

Identification of hub genes in colon cancer via bioinformatics analysis.

OBJECTIVES: This study aimed to investigate hub genes and their prognostic value in colon cancer via bioinformatics analysis. METHODS: Differentially expressed genes (DEGs) of expression profiles (GSE33113, GSE20916, and GSE37364) obtained from Gene Expression Omnibus (GEO) were identified using the GEO2R tool and Venn diagram software. Function and pathway enrichment analyses were performed, and a protein-protein interaction (PPI) network was constructed. Hub genes were verified based on The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) databases. RESULTS: We identified 207 DEGs, 62 upregulated and 145 downregulated genes, enriched in Gene Ontology terms "organic anion transport," "extracellular matrix," and "receptor ligand activity", and in the Kyoto Encyclopedia of Genes and Genomes pathway "cytokine-cytokine receptor interaction." The PPI network was constructed and nine hub genes were selected by survival analysis and expression validation. We verified these genes in the TCGA database and selected three potential predictors (ZG16, TIMP1, and BGN) that met the independent predictive criteria. TIMP1 and BGN were upregulated in patients with a high cancer risk, whereas ZG16 was downregulated. The immunostaining results from HPA supported these findings. CONCLUSION: This study indicates that these hub genes may be promising prognostic indicators or therapeutic targets for colon cancer.

Microbial Profiles and Risk Factors of Preexisting Biliary Infection in Patients with Therapeutic Endoscopy.

BACKGROUND: The bile infection may already exist before the administration of an interventional procedure, despite no clinical manifestations of cholangitis detected. Blood cultures remained negative even in more than half of the febrile cases with cholangitis. Risk factors associated with bacterial growth in bile before the intervention are not well defined. To establish the bacterial profiles isolated from the bile samples and to identify risk factors for bacterial colonization in the bile system. METHODS: Individuals who underwent endoscopic retrograde cholangiopancreatography (ERCP) interventions were enrolled. Bile samples were aspirated and were immediately transferred into a sterile tube for storage. RESULTS: Positive bile cultures were detected in 363 (38.0%) of 956 patients, including 322 benign diseases and 41 malignances. Of 363 positive cases, 351 (96.7%) were monoinfection and 12 (3.3%) multi-infection. Escherichia coli were the most common Gram-negative bacteria (210, 56.0%), followed by Klebsiella pneumoniae (45, 12.0%). Enterococcus faecalis represented the most common Gram-positive microorganism (19, 5.07%), while Candida albicans (11, 2.93%) were the dominant fungi. Klebsiella pneumoniae were more frequently detected in malignant diseases (P = 0.046). Age, previous ERCP history or OLT history, and CBD diameter were independent risk factors for positive cultures (P < 0.05) while preoperative jaundice drug therapy was the protective factor for bile infection (P < 0.05). CONCLUSION: Monomicrobial infection was dominant among all infections, and Klebsiella pneumoniae strains were more frequently isolated from patients with malignant diseases. To effectively manage patients who are at a high risk for bile infection, a detailed diagnosis and treatment plan for each case should be prepared.

Sphingosine kinase 1 promotes the metastasis of colorectal cancer by inducing the epithelial­mesenchymal transition mediated by the FAK/AKT/MMPs axis.

It was demonstrated that Sphingosine kinase 1 (SphK1) promotes tumor progression and confers the malignancy phenotype of colorectal cancer by activating the focal adhesion kinase (FAK) pathway. However, further clarification is required to determine if SphK1 promotes the metastasis of colorectal cancer by inducing epithelial­mesenchymal transition (EMT), and its mechanisms have not been fully elucidated. Immunohistochemistry staining was used to detect protein expression in normal colonic mucosa tissues and colorectal cancer tissues. Cells were transfected to overexpress SphK1, downregulate SphK1 or downregulate FAK. An MTT assay was used to detect the drug toxicity to cells. Transwell and wound healing assays were used to detect cell migration ability. Reverse transcription­polymerase chain reaction and western blot analysis were used to detect the expression of mRNA and protein, respectively. Scanning electron microscopy was used to observe the microvilli and pseudopodia of the cells. The analysis of protein expression in 114 human colorectal cancer tissues demonstrated that the expressions of SphK1, FAK, phosphorylated (p)­FAK, E­cadherin and vimentin were associated with the metastasis of colorectal cancer. Furthermore, the patients with colorectal cancer with SphK1­positive cancer demonstrated poorer prognosis compared with SphK1­negative cancer. FAK knockdown and SphK1 knockdown of human colon cancer RKO cells inhibited the EMT and migrational potency, along with the expression of p­FAK, p­protein kinase B (AKT) and matrix metalloproteinase (MMP)2/9. In contrast, SphK1 overexpression promoted EMT, migrational potency, and the expression of p­FAK, p­AKT and MMP2/9 in HT29 cells. Additionally, the EMT and migrational potency of SphK1­overexpressing HT29 cells was suppressed by a FAK inhibitor, and the expression of p­FAK, p­AKT and MMP2/9 was suppressed by blocking the FAK pathway. In conclusion, SphK1 promoted the migration and metastasis of colon cancer by inducing EMT mediated by the FAK/AKT/MMPs axis.

Role of Wnt/ß-catenin pathway agonist SKL2001 in Caerulein-induced acute pancreatitis.

The goal of this study was to clarify the protective role of the Wnt/ß-catenin pathway agonist SKL2001 in a rat model of Caerulein-induced acute pancreatitis. AR42J cells and rats were divided into 4 groups: control, Caerulein, SKL2001 + Caerulein, and SKL2001 + control. Cell apoptosis was examined using flow cytometry. Hematoxylin-eosin staining was performed to observe pathological changes in pancreatic and small intestinal tissues. Inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA), while genes related to the Wnt/ß-catenin pathway were quantified using quantitative real-time PCR. In vitro results showed that Caerulein promoted cell necrosis, inhibited the Wnt/ß-catenin pathway, and increased the level of inflammatory cytokines. However, SKL2001 reduced cell necrosis and inflammatory cytokines and activated the Wnt/ß-catenin pathway. Additionally, in vivo results demonstrated the accumulation of fluid (i.e., edema), hemorrhage, inflammation and necrosis of the pancreatic acini occurred 6 h after the final Caerulein induction, with the damage reaching a maximal level 12 h after the final Caerulein induction; meanwhile, the Wnt/ß-catenin pathway was evidently inhibited with an enhanced level of inflammatory cytokines. The aforementioned damage was further aggravated 12 h later. Nevertheless, the pancreatic and small intestinal tissue damages were alleviated in Caerulein-induced rats treated with SKL2001. In conclusion, activation of the Wnt/ß-catenin pathway could inhibit Caerulein-induced cell apoptosis and inflammatory cytokine release, thus improving pancreatic and intestinal damage in rats with acute pancreatitis.

NIK­ and IKKß­binding protein contributes to gastric cancer chemoresistance by promoting epithelial­mesenchymal transition through the NF­κB signaling pathway.

Systematic chemotherapy is indispensable for gastric cancer patients with advanced stage disease, but the occurrence of chemoresistance drastically limits treatment effectiveness. There is a tremendous need for identifying the underlying mechanism of chemoresistance. NIK­ and IKKß­binding protein (NIBP) (also known as TRAPPC9, trafficking protein particle complex 9) is a regulator of the cytokine­induced NF­κB signaling pathway which has been proven to play pivotal roles in the progression of various malignancies. Nevertheless, it is still ambiguous whether NIBP is involved in the chemoresistance of gastric cancer. The aim of the present study was to investigate the effect of NIBP on chemotherapy resistance of gastric cancer (GC) and to research the mechanisms of Ginkgo biloba extract 761 (EGb 761®) on reversing chemoresistence which has been confirmed in our previous study. In the present study, the results of immumohistochemisty revealed that the positive staining rates of NIBP, NF­κB p65 and NF­κB p­p65 in gastric cancer tissues were obviously higher than those in normal tissues. Furthermore, a close correlation was found to exist between the expression of NIBP and NF­κB p65 (p­p65) in gastric cancer tissues. Moreover, the overexpression of NIBP was closely related to tumor differentiation, depth of invasion, clinical stage and lymphatic metastasis in gastric cancer. Western blot analysis, real­time PCR, MTT assay and flow cytometric analysis were performed and the results demonstrated that compared with the gastric cancer SGC­7901 cells, the expression of NIBP, NF­κB p65, NF­κB p­p65 and mesenchymal marker vimentin were significantly increased in gastric cancer multidrug­resistant SGC­7901/CDDP cells, and the epithelial cell marker ZO­1 was significantly decreased. Meanwhile, it was found that SGC­7901/CDDP cells were accompanied by spindle­like mesenchymal appearance and upregulation of stem cell marker CD133 which has been verified to be an upstream regulatory gene of epithelial­mesenchymal transition (EMT). Further research confirmed that downregulation of NIBP by Ginkgo biloba extract (EGb) 761 EGb 761 suppressed the cis­diamminedichloroplatinum(II) (CDDP)­induced NF­κB signaling pathway, EMT and the expression of CD133 in SGC­7901 and SGC­7901/CDDP cells. Altogether, these data indicate that the NIBP­regulated NF­κB signaling pathway plays a pivotal role in the chemoresistance of gastric cancer by promoting CD133­induced EMT.

SphK1 modulates cell migration and EMT-related marker expression by regulating the expression of p-FAK in colorectal cancer cells.

Sphingosine kinase 1 (SphK1) plays an important role in colorectal carcinoma metastasis. However, whether SphK1 modulates epithelial-mesenchymal transition (EMT)-related marker expression and the underlying mechanisms remain unclear. In this study, in order to clarify this issue, we used various colorectal cancer (CRC) cell lines, Caco2, HT29, RKO and HCT116. Each of the cell lines was divided into 3 groups as follows: the control group, SKI-Ⅱ (SphK1 inhibitor) group and PF-562271 [focal adhesion kinase (FAK) inhibitor] group. The migratory ability of the cells was examined by Transwell chamber assay. The mRNA and protein expression levels of SphK1, FAK (p-FAK), Slug, vimentin, N-cadherin and E-cadherin were detected by PCR and western blot analysis, respectively. The results revealed that the suppression of SphK1 reduced the cell migratory ability, and decreased the expression of Slug, vimentin and N-cadherin; however, the expression of E-cadherin was increased. Moreover, the inhibition of SphK1 reduced the expression of p-FAK. The inhibition of FAK (p-FAK) also decreased the cell migratory ability, and decreased the expression of Slug, vimentin and N-cadherin, whereas the expression of E-cadherin was increased. Thus, our data suggest that SphK1 modulates the expression of EMT-related markers and cell migration by regulating the expression of p-FAK in CRC cells. Thus, SphK1 may play a functional role in mediating the EMT process in CRC.

NIBP impacts on the expression of E-cadherin, CD44 and vimentin in colon cancer via the NF-κB pathway.

NIBP, a novel nuclear factor-κB (NF-κB)-inducing kinase (NIK) and IκB kinase ß (IKKß) binding protein, directly interacts with NIK and IKKß, and acts as the 'bridge' of the NF­κB classical and alternative signaling pathways. However, its influence on epithelial­mesenchymal transition markers in colon cancer remains to be fully elucidated. The aim of the present study was to investigate the roles of NIBP impacting on the expression of E­cadherin, CD44 and vimentin. In the present study, the associations between NIBP and E­cadherin, CD44 and vimentin in clinical samples were analyzed by making pairwise comparisons between normal colon tissue, non­metastatic colon cancer tissue and metastatic colon cancer tissue. In in vitro experiments, after changing the expression of NIBP in cells, the protein expression levels of CD44, vimentin, E­cadherin were analyzed by western blot analysis. The results revealed that the protein expression levels of NIBP, CD44 and vimentin were markedly increased, and E­cadherin was markedly decreased, in metastatic colon cancer tissue compared with normal colon tissue and non­metastatic colon cancer tissue. Upregulation of NIBP expression decreased the levels of E­cadherin, whereas the downregulation of NIBP increased E­cadherin levels, while no significant differences were observed in the levels of CD44 and vimentin. In addition, cells that were treated with the NF­κB inhibitor, pyrrolidine dithiocarbamate (PDTC), also tended to exhibit increased levels of CD44 and vimentin expression in the NIBP upregulated expression group (29­NIBP group) compared with the mock group, whereas the expression levels of E­cadherin, CD44 and vimentin were similar in the NIBP downregulated expression group (116­NIBPmir group) and the HCT116 blank control group (116­mock group) on treatment of the cells with tumor necrosis factor­α. These findings indicated that NIBP, E­cadherin, CD44 and vimentin are possibly associated with metastasis in colon cancer. When the NF­κB pathway is not subjected to any interventions, NIBP may predominantly regulate the NF­κB classical pathway, rather than the alternative pathway. When the classical pathway was completely inhibited, NIBP was able to activate the NF­κB alternative pathway. NIBP is therefore necessary for the interaction between the NF­κB classical and alternative pathways. In conclusion, NIBP impacts on the expression levels of E­cadherin, CD44 and vimentin via the NF­κB classical and alternative pathways. Therapeutic regimens for patients with colorectal cancer may comprise NIBP inhibitors in the future.

Ginkgo biloba extract enhances chemotherapy sensitivity and reverses chemoresistance through suppression of the KSR1-mediated ERK1/2 pathway in gastric cancer cells.

Kinase suppressor of Ras 1 (KSR1) is a scaffold protein that modulates the activation of the oncogenic mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway. Ginkgo biloba extract (EGb) 761 has been demonstrated to possess antitumor activity that may be related to the KSR1-mediated ERK signaling pathway. However, the roles and its underlying mechanism in gastric cancer are unclear. In the present study, 62 gastric cancer and matched normal tissues were exploited for immunohistochemistry and real-time fluorescent quantitative PCR detection. Results of the immunohistochemistry showed that the expression of ERK1/2 and p-ERK1/2 was correlated to the expression of KSR1 and p-KSR1 in the gastric cancer tissues, and the overexpression of KSR1, p-KSR1, ERK1/2 and p-ERK1/2 was significantly associated with histological grade, TNM stage, lymph node and distant metastasis. Compared with the normal tissues, the relative mRNA copy values of KSR1, ERK1 and ERK2 in the cancer tissues were 2.43 ± 0.49, 2.10 ± 0.44 and 3.65 ± 0.94. In addition, the expression of KSR1, p-KSR1, ERK1/2 and p-ERK1/2 in human gastric cancer multidrug resistant SGC-7901/CDDP cells was higher than that in the SGC-7901 cells as detected by the methods of immunocytochemistry and western blot analysis. EGb 761 not only suppressed expression of these proteins induced by cisplatin (CDDP) and etoposide in SGC-7901 cells, but also inhibited expression of these proteins in the SGC-7901/CDDP cells. Meanwhile, the proliferation-suppressing and apoptosis-inducing capacities of CDDP and etoposide were enhanced following combined treatment with EGb 761. Moreover, EGb 761 reduced the malondialdehyde (MDA) content and elevated the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the tumor cells. These results confirmed that activation of the KSR1-mediated ERK1/2 signaling pathway may contribute to tumorigenesis, metastasis and chemoresistance of human gastric cancer. EGb 761 enhanced the chemotherapy sensitivity and reversed the chemoresistance through suppression of the KSR1-mediated ERK1/2 pathway in gastric cancer cells, and the underlying mechanism may be related to its antioxidative activity.

Effect of trichostatin A on CNE2 nasopharyngeal carcinoma cells--genome-wide DNA methylation alteration.

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor. We here investigated its effects on proliferation and apoptosis of the CNE2 carcinoma cell line, and attempted to establish genome-wide DNA methylation alteration due to differentially histone acetylation status. After cells were treated by TSA, the inhibitory rate of cell proliferation was examined with a CCK8 kit, and cell apoptosis was determined by flow cytometry. Compared to control, TSA inhibited CNE2 cell growth and induced apoptosis. Furthermore, TSA was found to induce genome-wide methylation alteration as assessed by genome-wide methylation array. Overall DNA methylation level of cells treated with TSA was higher than in controls. Function and pathway analysis revealed that many genes with methylation alteration were involved in key biological roles, such as apoptosis and cell proliferation. Three genes (DAP3, HSPB1 and CLDN) were independently confirmed by quantitative real-time PCR. Finally, we conclude that TSA inhibits CNE2 cell growth and induces apoptosis in vitro involving genome-wide DNA methylation alteration, so that it has promising application prospects in treatment of NPC in vivo. Although many unreported hypermethylated/hypomethylated genes should be further analyzed and validated, the pointers to new biomarkers and therapeutic strategies in the treatment of NPC should be stressed.

Melatonin reduces inflammation and recovers endogenous ghrelin in acute necrotizing pancreatitis in rats.

The present study aimed to evaluate the therapeutic effects of melatonin on either ghrelin secretion or gastric mucosal injury in acute necrotizing pancreatitis (ANP). ANP was induced in rats by L-arginine. Prior to L-arginine injection, the rats were pre-treated with melatonin for 30 min. Following the last injection, the animals were sacrificed at different time-points. The levels of ghrelin and melatonin in the serum and gastric tissue were detected by ELISA. Levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and malondialdehyde (MDA) as well as total superoxide dismutase (T-SOD) activities in gastric tissue were measured. In rats with ANP, acute gastric injury was observed, and the levels of MDA, SOD, TNF-α and IL-6 were significantly increased. The melatonin levels in serum or gastric tissue peaked at 6 h and returned to normal levels at 12 h after melatonin was administered. However, ghrelin remained at low levels during the first 12 h, but it recovered at 24 h and continued increasing, while the levels of oxidative stress damage and activity of inflammatory factors were decreased. The protective effects of melatonin on acute gastric injury during the early stages of ANP may be mediated through anti-oxidative and anti-inflammatory activities, while at advanced stages of ANP, it may be mediated through the recovered endogenous ghrelin.

[Sphingosine kinase 1 enhances the proliferation and invasion of human colon cancer LoVo cells through up-regulating FAK pathway and the expression of ICAM-1 and VCAM-1].

OBJECTIVE: To investigate the effects of sphingosine kinase 1 (SphK1) on the proliferation, migration and invasion of human colon cancer LoVo cells, and to explore the related mechanisms. METHODS: Human colon cancer LoVo cells were divided into three groups: phorbol 12-myristate 13-acetate (PMA) was used to induce the activation of SphK1 in the PMA group, N,N-dimethylsphingosine (DMS) used to suppress the activity of SphK1 in DMS group, and the cells treated with equal amount of 0.9 % NaCl instead of drugs served as the control group. The activity of SphK1 was assayed by autoradiography, the cell proliferation was assessed by MTT assay, cell migration and invasion were examined by Boyden chamber assay, concentrations of sICAM-1 and sVCAM-1 were assayed by ELISA, and RT-PCR and Western blot were used to evaluate the mRNA and protein expression in the cells. RESULTS: The activity of SphK1 was efficiently induced by PMA and significantly suppressed by DMS. PMA induced cell proliferation in a time- and dose-dependent manner. On the contrast, DMS suppressed cell proliferation in a time- and dose-dependent manner. After treating with PMA, the number of migrating and invasing cells were increased to 143.36 ± 8.73 and 118.46 ± 6.25, significantly higher than those of the control group (75.48 ± 6.12 and 64.19 ± 5.36). After treating with DMS, the number of migrating and invasing cells were decreased to 38.57 ± 3.24 and 32.48 ± 4.27, significantly lower than those of the control group (P < 0.01). The relative expression levels of FAK, ICAM-1 and VCAM-1 mRNA in the PMA group were 0.82 ± 0.06, 0.74 ± 0.05 and 0.89 ± 0.09, and those in the DMS group were 0.23 ± 0.02, 0.26 ± 0.03 and 0.37 ± 0.04, with significant differences between the PMA, DMS and control groups (P < 0.01). Compared with the control group, the relative expression levels of FAK and p-FAK proteins in the PMA group (0.52 ± 0.06 and 0.51 ± 0.06) were significantly elevated, and those of the DMS group (0.20 ± 0.03 and 0.09 ± 0.02) were significantly decreased. In addition, the concentrations of sICAM-1 and sVCAM-1 were significantly elevated with the activation of SphK1. On the contrary, those of the DMS group were significantly reduced with the suppression of SphK1 (Both P < 0.01). CONCLUSIONS: SphK1 may enhance the proliferation, migration and invasion of colon cancer LoVo cells through activating FAK pathway and up-regulating the expression of ICAM-1 and VCAM-1.

Sphingosine kinase 1 promotes tumor progression and confers malignancy phenotypes of colon cancer by regulating the focal adhesion kinase pathway and adhesion molecules.

Studies suggest a tumor-promoting function of sphingosine kinase 1 (SphK1) in some types of human tumors, however, its effect on colon cancer is still unclear. The aims of this study were to investigate the roles of SphK1 in the progression and tumor cell phenotypic changes in colon cancer. Moreover, the focal adhesion kinase (FAK) pathway and the expression of intercellular adhesion molecule­1 (ICAM­1) and vascular cell adhesion molecule­1 (VCAM­1) were detected to explore the mechanisms of SphK1 action. In this study, the expression of SphK1, FAK and phospho-FAK (p-FAK) was analyzed in 66 surgical specimens of primary colon cancer and matched adjacent normal tissues by immunohistochemistry and western blotting. In addition, N,N-dimethylsphingosine (DMS), SphK1 DNA and shRNA transfection were used to regulate the expression and activity of SphK1 in the LOVO colon cancer cell line. Tumor cell phenotypic changes were analyzed by cell viability, invasion and apoptosis assays. Results showed that the expression of SphK1, FAK and p-FAK in colon cancer tissues were significantly stronger compared to those in matched normal tissues. There was a close correlation between the expression of SphK1 and FAK or p-FAK and the co-expression of SphK1, FAK and p-FAK significantly associated with histological grade, Dukes' stage, lymph node metastasis and distant metastasis. Overexpression of SphK1 after DNA transfection enhanced tumor cell viability and invasiveness, but suppressed cell apoptosis. In contrast, suppression of SphK1 by DMS and shRNA reduced tumor cell viability and invasiveness, but promoted cell apoptosis. The expression of FAK, p-FAK, ICAM-1 and VCAM-1 in LOVO cells were increased with the overexpression of SphK1 but decreased with the suppression of SphK1. These findings indicate that SphK1 regulates tumor cell proliferation, apoptosis and invasion, which ultimately contributes to tumor progression and malignancy phenotype in colon cancer. FAK pathway, ICAM-1 and VCAM-1 may play critical roles in this SphK1­mediated effect.

Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and uPA via MAPK pathways.

PURPOSE: Sphingosine kinase (SphK) 1 is an oncogenic enzyme promoting transformation, proliferation, and survival of a number of human tumor cells. However, its effect on colon cancer cell behavior has not been fully clarified. METHODS: SphK1 plasmid or SphK1 shRNA transfection and N,N-dimethylsphingosine (DMS) was used to regulate the expression and activity of SphK1 in colon cancer line LOVO. Cell proliferation, apoptosis, invasion, and protein expression were detected by MTT, flow cytometry, transwell chambers model, and western blot. The levels of metalloproteinases-2/9 (MMP-2/9) and urokinase plasminogen activator (uPA) were detected by ELISA. RESULTS: Overexpression of SphK1 after plasmid transfection markedly enhanced LOVO cell viability and invasiveness and reduced cell apoptosis. In contrast, inhibition of SphK1 by DMS and shRNA significantly suppressed cell viability and invasiveness but promoted cell apoptosis. SphK1 increased the constitutive expression of extracellular signal-regulated kinase1/2 (ERK1/2) but reduced the constitutive expression of p38 mitogen-activated protein kinase (MAPK). Blocking ERK1/2 pathway inhibited the biological effects induced by overexpression of SphK1. Blocking p38 MAPK pathway reversed the effects of DMS and SphK1 shRNA. Moreover, SphK1 was required for the production of MMP-2/9 and uPA in tumor cells, which was suppressed by ERK1/2 inhibitor U0126, but enhanced by the p38 MAPK inhibitor SB203580. CONCLUSIONS: SphK1 enhances colon cancer cell proliferation and invasiveness, meanwhile suppressing cell apoptosis. SphK1 promoting the secretion of MMP-2/9 and uPA via activation of ERK1/2 and suppression of p38 MAPK pathways maybe the molecular mechanisms for its regulation of the malignant behavior of colon cancer cell.

[Effect of sphingosine kinase 1 on the apoptosis, migration and invasion of colon cancer HT-29 cells and its molecular mechanisms].

OBJECTIVE: To investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms. METHODS: Phorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK. RESULTS: PMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced. CONCLUSIONS: SphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.

Inhibition of SPHK1 suppresses phorbol 12-myristate 13-acetate-induced metastatic phenotype in colorectal cancer HT-29 cells.

Expression of sphingosine kinase 1 (SPHK1) plays a role in colorectal cancer progression. This study aimed to demonstrate the mechanism of human colorectal cancer cell metastatic phenotype through SPHK1 knockdown. Human colorectal cancer HT-29 cells were stimulated by phorbol 12-myristate 13-acetate (PMA) with or without SPHK1 siRNA transfection. Tumor cell phenotypic changes were analyzed by using invasion, motility, cell viability, and apoptosis assays. Gene expressions were assessed using Western blot. PMA induced a metastatic phenotype in colorectal cancer cells, as indicated by cell viability, migration and invasion capacity, and ERK1/2 phosphorylation, whereas SPHK1 siRNA transfection suppressed the metastatic phenotype of tumor cells and antagonized PMA's effects. SPHK1 knockdown also inhibited secretion of MMP-2 and MMP-9 into the tumor cell conditioned medium. Suppression of SPHK1 expression suppresses the PMA-induced metastatic phenotype via ERK1/2 phosphorylation in human colorectal cancer cells.