Jie-An Huang

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

PurposeSphingosine 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.MethodsSphK1 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.ResultsOverexpression 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.ConclusionsSphK1 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.

NIK- and IKKβ-binding protein promotes colon cancer metastasis by activating the classical NF-κB pathway and MMPs

The nuclear factor (NF)-κB pathway has been implicated in colorectal cancer (CRC) tumorigenesis. Here, we investigated the role of a novel NIK- and IKKβ-binding protein (NIBP) in CRC metastasis through activation of the canonical NF-κB pathway. NIBP, p-p65, and matrix metalloproteases (MMPs) were assessed by immunohistochemistry in 114 CRC tissues, and the time to metastasis was recorded after surgery. Furthermore, the activity of the NF-κB pathway, MMP expression, and the metastatic potential of HT-29 cells overexpressing NIBP after treatment with the NF-κB inhibitor pyrrolidinecarbodithioic acid (PDTC) were examined in vitro and in vivo. NIBP-positive CRC exhibited a higher rate of metastasis, and the time to metastasis of NIBP-positive patients was shorter in the early tumor, lymph node, metastasis (TNM) stages (I and II), while NIBP and p-p65 expression was higher in later TNM stages (III and IV). However, there was no difference in terms of the positive rate of NIBP, p-p65, MMP-2, and serum carcinoembryonic antigen (CEA) level was no difference in the pathological type, gender, tumor location, or size. The NF-κB pathway, MMP-2 and MMP-9 activity, and cell motility and invasion were increased in NIBP-overexpressing cells, even after PDTC treatment. Moreover, these cells exhibited high metastasis in mice, and p-p65, MMP-2, and MMP-9 expression levels were elevated in the primary tumor and liver metastases. In conclusion, NIBP overexpression increases the CRC metastatic potential through activation of the NF-κB pathway and increasing MMP-2 and MMP-9 expression. In addition, NIBP overexpression, at least in part, may reduce inhibition of the canonical NF-κB pathway and MMPs caused by PDTC treatment.

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.

Knockdown of NIK and IKKβ-Binding Protein (NIBP) Reduces Colorectal Cancer Metastasis through Down-Regulation of the Canonical NF-κΒ Signaling Pathway and Suppression of MAPK Signaling Mediated through ERK and JNK.

Background Despite the identification of many signaling pathways involved in colorectal cancer (CRC) tumorigenesis, metastatic CRC still remains one of the major causes of cancer related death. NIK and IKKβ-binding protein (NIBP) is one of the key regulators of the NF-κB signaling pathway, which has been implicated in CRC metastasis. The aim of this study was to investigate the possible role of NIBP in CRC metastasis through its regulation of NF-κΒ and extracellular regulated kinase/c-Janus kinase (ERK/JNK) signaling pathways. Methods In this study NIBP, phosphorylated (p)-p65, p-ERK1/2, and p-JNK1/2 expression was examined in 130 CRC, and 25 adenoma tissue samples were studied by immunohistochemistry. NIBP shRNA knockdown was performed in HCT116 cells, and NF-κB and ERK/JNK pathway activity was measured after TNF-α stimulation in vitro and in vivo. Results We found that NIBP, p-p65, p-ERK1/2, and p-JNK1/2 expression was higher in late stages of CRC compared to early stages or adenomas. Expression of p-p65, p-IκBα, p-IκBβ, p-ERK1/2, and p-JNK1/2 was inhibited in TNF-α stimulated HCT116 cells following NIBP knockdown. Nevertheless, p-ERK1/2 expression in un-transfected and NIBP knockdown HCT116 cells remained the same in the absence of TNF-α stimulation. Furthermore, cell motility and invasion were reduced in HCT116 cells following NIBP knockdown even after TNF-α treatment. Finally, primary tumor weight and liver metastasis were reduced in nude mice with orthotopically transplanted NIBP knockdown of HCT116 cells. Conclusion In conclusion, we demonstrated that NIBP knockdown reduces colorectal cancer metastasis through down-regulation of canonical NF-κΒ signaling and suppression of ERK and JNK signaling.

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 PMAs 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.

Oxymatrine inhibits epithelial-mesenchymal transition through regulation of NF-κB signaling in colorectal cancer cells.

Oxymatrine, a traditional Chinese herb extracted from Sophora flavescens Ait., displays strong anti-inflammatory and anticancer activities, but how oxymatrine exhibits anticarcinogenic effects in human colorectal cancer (CRC) remains uncertain. The present study aimed to elucidate the exact mechanism by which oxymatrine exhibits anticarcinogenic effects in CRC using the human colon cancer RKO cell line as the experimental model. CRC cells were treated with oxymatrine, and cell proliferation, migration and invasion were examined by colorimetric MTT, Transwell chamber and wound healing assays, respectively. In addition, epithelial-mesenchymal transition (EMT) markers and p65 were assessed by western blot analysis. Our study demonstrated that oxymatrine hindered the proliferation, migration and invasion of the CRC cells. Mechanistically, we found that oxymatrine modulated the expression of EMT markers including E-cadherin, Snail and N-cadherin, and reduced expression of p65 which is crucial to NF-κB activation. In conclusion, our results indicate that oxymatrine reduces the activation of the NF-κB signaling pathway and inhibits CRC invasion by modulating EMT.

Activation of the SphK1/ERK/p‑ERK pathway promotes autophagy in colon cancer cells

Sphingosine kinase 1 (SphK1) is a master kinase that catalyzes the synthesis of sphingosine 1 phosphate and participates in the regulation of cell proliferation and autophagy. The present study aimed to assess the effects of the activation of the SphK1/extracellular signal-regulated kinase (ERK)/phosphorylated (p-)ERK pathway in the regulation of autophagy in colon cancer (HT-29) cells. Inverted fluorescence microscopy was used to detect the expression of green fluorescent protein (GFP) in the SphK1-overexpressing HT-29 cells [SphK1(+)-HT-29] and the negative control HT-29 cells (NC-HT-29). Western blotting was used to detect the protein expression levels of SphK1, ERK1/2, p-ERK1/2, as well as those of the autophagy-associated markers LC3A, ATG5, and ULK1. Protein localization and expression of the LC3A antibody were detected by immunofluorescence. The results demonstrated that GFP was similarly expressed in SphK1(+)-HT-29 and NC-HT-29 cells. However, significantly increased SphK1 mRNA and protein expression levels were detected in SphK1(+)-HT-29 cells compared with in NC-HT-29 cells, which resulted in upregulated ERK/p-ERK. Furthermore, the protein expression levels of the three autophagy-associated markers increased. LC3A protein was localized in the cytoplasm of SphK1(+)-HT-29 cells, indicating autophagy. In summary, the findings of the present study suggested that activation of the SphK1/ERK/p-ERK pathway promotes autophagy in colon cancer HT-29 cells.