Jianhui Fan

Curcumin Inhibits the Migration and Invasion of Mouse Hepatoma Hca-F Cells Through Down-regulating Caveolin-1 Expression and Epidermal Growth Factor Receptor Signaling

Mouse hepatoma cellular carinoma cell line (Hca‐F) cells have highly invasive and lymphatic metastasis potential in vitro and in vivo. Curcumin (diferuloylmethane) is an active component of the spice turmeric and has a diversity of antitumor activities. However, there is no available information to address the effects of curcumin on migration and invasion of mouse hepatoma Hca‐F cells. In this study, we found that curcumin exerted a concentration‐ and time‐dependent inhibitory effect on the migration and invasion of Hca‐F cells in vitro. Curcumin inhibited the expression of the tumor promoter caveolin‐1 (Cav‐1) in Hca‐F cells. Up‐regulation of Cav‐1 expression by pcDNA3.1/Cav‐1 plasmid was able to reverse the curcumin‐induced antimigration and anti‐invasion effects in vitro. Curcumin down‐regulated the expression of cluster of differntiation (CD)147, matrix metalloproteinase 2, and matrix metalloproteinase 9 and inhibited the phosphorylation of epidermal growth factor receptor (EGFR), the phosphoinositilde 3‐kinase (PI3K)/protein kinase B (Akt), p38 mitogen‐activated protein kinase (MAPK), and p44/42MAPK in Hca‐F cells. Taken together, our findings suggest that curcumin can suppress the migratory and invasive ability of mouse hepatoma Hca‐F cells, and this action is mediated through a novel mechanism involving inactivation of Cav‐1 and EGFR signaling pathways.

MiR-34a inhibits lymphatic metastasis potential of mouse hepatoma cells

MicroRNAs are small non-coding RNAs that regulate the expression of other genes in a post-transcriptional manner. MiR-34a can induce apoptosis, cell cycle arrest, and senescence. However, its role in tumor progress remains to be fully elucidated. In the present study, the role of miR-34a in lymphatic metastasis was investigated using mouse hepatocarcinoma cell lines Hca-F and Hepa1-6. MicroRNA profiling and Hairpin-RT-PCR analysis showed that the expression level of miR-34a was higher in Hepa1-6 cells (of no metastatic ability) than that in Hca-F cells (of high metastatic ability). Ectopic expression of miR-34a can inhibit cell growth and cell invasion in Hepa1-6 and Hca-F cells. Moreover, miR-34a triggers G1 arrest and down-regulates CyclinD1 and CDK6 in Hepa1-6 cells. Furthermore, we proved that miR-34a decreased adhesion of Hca-F cells to regional lymph node in vitro, reduced lymph nodes-metastasized burden, and inhibited tumor lymph node metastases in vivo. All these results suggest that miR-34a plays multiple tumor suppressive roles in murine hepatocarcinoma, not only inhibiting cell growth by cell cycle arrest, but also repressing metastasis, and may serve as a novel therapeutic target for hepatocarcinoma.

N-acetylglucosaminyltransferase IVa regulates metastatic potential of mouse hepatocarcinoma cells through glycosylation of CD147

N-acetylglucosaminyltransferase (GnT)-IV a is a key enzyme that catalyzes the formation of the GlcNAC β1-4 branch on the core structure of complex N-Glycans, which is the common substrate for other N-acetylglucosaminyltransferases, such as GnT-III and GnT-V. Our recent study indicates that the expression of GnT-IVa in Hca-F cells was much higher than that in Hepa1-6 cells, these two mouse hepatocarcinoma cell lines have high and no metastatic potential in lymph nodes respectively. To investigate the effects of GnT-IVa on the metastasis of hepatocarcinoma, exogenous GnT-IVa was introduced into Hepa1-6 cells, and on the other hand, the expression of GnT-IVa was down-regulated in Hca-F cells. The engineered overexpression of GnT-IVa in Hepa1-6 cells increased the antennary branches of complex N-glycans and reduced bisecting branches in vitro and in vivo, which leads to the increase in migration and metastatic capability of hepatocarcinoma cells. Conversely, down-regulated expression of GnT-IVa in Hca-F cells showed reduced tetra-antennary branches of N-Glycans, and significantly decreased the migration and metastatic capability. Furthermore, we found that the regulated GnT-IVa converts the heterogeneous N-glycosylated forms of CD147 in Hepa1-6 and Hca-F cells, and significantly changed the antennary oligosaccharide structures on CD147. These results suggest that GnT-IVa could be acting as a key role in migration and metastasis of mouse hepatocarcinoma cells through altering the glycosylation of CD147. These findings should be valuable in delineating the important function of GnT-IVa during the process of hepatocarcinoma growth and metastasis.

Caveolin-1 up-regulates integrin α2,6-sialylation to promote integrin α5β1-dependent hepatocarcinoma cell adhesion.

The alterations of integrin glycosylation play a crucial role in tumor metastasis. Our previous studies indicated that caveolin‐1 promoted the expression of the key α2,6‐sialytransferase ST6Gal‐I and fibronectin‐mediated adhesion of mouse hepatocarcinoma cell. Herein, we investigated the role of α2,6‐sialylated α5‐integrin in the adhesion of mouse hepatocarcinoma H22 cell. We demonstrated that caveolin‐1 up‐regulated cell surface α2,6‐linked sialic acid via stimulating ST6Gal‐I transcription. Cell surface α2,6‐sialylation was required for integrin α5β1‐dependent cell adhesion to fibronectin, and an increase in α2,6‐linked sialic acid on α5‐subunit facilitated fibronectin‐mediated focal adhesion kinase phosphorylations, suggesting that α2,6‐sialylated α5‐subunit promoted integrin α5β1‐dependent cell adhesion.

Caveolin-1 up-regulates ST6Gal-I to promote the adhesive capability of mouse hepatocarcinoma cells to fibronectin via FAK-mediated adhesion signaling.

Caveolin-1 is a major structural protein of caveolae and plays important functions in tumorigenesis and development. Hca-F and Hepa1-6 are mouse hepatocarcinoma cell lines with high and low malignant potential, respectively. Our previous studies revealed that caveolin-1 promoted cell invasion by up-regulating the glycosylation of matrix metalloproteinase inducer CD147 of Hepa1-6 and Hca-F cells. However, the roles of caveolin-1 in cell-ECM adhesion and the mechanisms involved remain unknown. This study showed that caveolin-1 overexpression in Hepa1-6 cells up-regulated sialyltransferase ST6Gal-I expression and activated FAK-mediated adhesion signaling, and down-regulation of ST6Gal-I attenuated caveolin-1-induced increase in the adhesive ability of Hepa1-6 cells to fibronectin. Conversely, caveolin-1 knockdown in Hca-F cells inhibited ST6Gal-I expression and FAK signaling-mediated cell adhesion to fibronectin. Re-expression of wild-type caveolin-1 or ST6Gal-I rescued the decreased ST6Gal-I expression and adhesion of Hca-F cells caused by caveolin-1 silencing. Further studies indicated that caveolin-1 might regulate ST6Gal-I expression through caveolin-1 scaffolding domain. Taken together, these results demonstrate for the first time that caveolin-1 can up-regulate ST6Gal-I expression and further contribute to promoting mouse hepatocarcinoma cell adhesion to fibronectin by activating FAK-mediated adhesion signaling.

Let-7c inhibits metastatic ability of mouse hepatocarcinoma cells via targeting mannoside acetylglucosaminyltransferase 4 isoenzyme A

Aberrant glycosylation may promote tumor invasion and metastasis. To investigate whether microRNA (miRNA) is involved in glycosylation-related metastasis, we examined the role of let-7c, a well-known tumor-suppressor miRNA, in glycosylation in murine hepatocarcinoma cell lines Hca-F and Hca-P. We found that let-7c level was higher in Hca-P cells (with lower lymphatic metastasis potential) than in Hca-F cells (with higher lymphatic metastasis potential). Overexpression of let-7c decreased hyper-N-glycosylation of Hca-F cells and repressed their metastatic and invasive ability. Mannoside acetylglucosaminyltransferase 4, isoenzyme A (Mgat4a) is a key glycosyltransferase in the pathway of synthesizing complex N-glycans. Bioinformatics analysis indicates that Mgat4a may be a target of let-7c, which has been verified by dual-luciferase reporter gene assay. Furthermore, the anti-metastatic effect of overexpressed let-7c is similar to that of Mgat4a siRNAs transfection. Hence, our results suggest that let-7c may inhibit the metastatic ability of Hca-F cells, at least partially, via repressing Mgat4a activity.

CD147 stimulates hepatoma cells escaping from immune surveillance of T cells by interaction with Cyclophilin A

T cells play an important role in tumor immune surveillance. CD147 is a member of immunoglobulin superfamily present on the surface of many tumor cells and mediates malignant cell behaviors. Cyclophilin A (CypA) is an intracellular protein promoting inflammation when released from cells. CypA is a natural ligand for CD147. In this study, CD147 specific short hairpin RNAs (shRNA) were transfected into murine hepatocellular carcinoma Hepa1-6 cells to assess the effects of CD147 on hepatoma cells escaping from immune surveillance of T cells. We found extracellular CypA stimulated cell proliferation through CD147 by activating ERK1/2 signaling pathway. Downregulation of CD147 expression on Hepa1-6 cells significantly suppressed tumor progression in vivo, and decreased cell viability when co-cultured with T cells in vitro. Importantly, knockdown of CD147 on Hepa1-6 cells resulted in significantly increased T cells chemotaxis induced by CypA both in vivo and in vitro. These findings provide novel mechanisms how tumor cells escaping from immune surveillance of T cells. We provide a potential therapy for hepatocellular carcinoma by targeting CD147 or CD147-CypA interactions.

Downregulated protein O-fucosyl transferase 1 (Pofut1) expression exerts antiproliferative and antiadhesive effects on hepatocytes by inhibiting Notch signalling.

The Notch signalling pathway is essential for proper cell growth and development. Many factors interact in the cellular context to bring about its effects. Critical to this process is the influence of Pofut1 whose function is to fucosylate Notch receptors and ligands on the cell surface for proper interaction. Of the three liver cell lines, HepG2, SMMC-7721 (hepatoma cell lines) and L-02 (normal fetal liver cell line) were investigated. In the current study, Pofut1 was silenced in L-02 due to its significantly high level of expression, (P<0.05) using transient SiRNA. Notch1, Cyclin D1 and p53 were significantly suppressed consequently. The effect on cell cycle, proliferation, adhesion and migration were investigated. Evidence adduced, indicate a general modification of cellular function. While proliferation and adhesion were significantly inhibited, the cell cycle arrest was obvious (P<0.05), migration was not affected. The effects seen are akin to those reported in previous studies in hepatoma cells mimicking some of the effects of notch1 silencing. Results of this study indicate a possible role of Pofut1 conferring a tumor suppressor role through the Notch signalling pathway.

GnT-V promotes chemosensitivity to gemcitabine in bladder cancer cells through β1,6 GlcNAc branch modification of human equilibrative nucleoside transporter 1

Human equilibrative nucleoside transporter 1 (hENT1) transports nucleoside analogue drugs across cellular membranes and is necessary for the uptake of many anti-tumor drugs. Gemcitabine is a frontline agent of chemotherapy for bladder cancer despite its limited efficacy due to chemoresistance, there is an acute need to decipher mechanisms underlying chemosensitivity to gemcitabinein in bladder cancer cells. Here we report a novel role for N-acetylglucosaminyltransferase V (GnT-V) in gemcitabine chemosensitivity. In this study, we found that GnT-V expression affected cell death rate to gemcitabine in different bladder cancer cells and down-regulation of GnT-V inhibited the gemcitabine sensitivity with time and dose dependent way in T24 cells. Moreover, mechanistic investigations showed that silencing GnT-V caused dramatic decrease of β1,6 GlcNAc structure on hENT1 leading to apparently decreased accumulation of hENT1 at plasma membrane, and therefore result in less uptake of gemcitabine in T24/shRNA cells. Together, our present study indicated that GnT-V enhances gemcitabine chemosensitivity via modulation of hENT1 N-glycosylation and transport activity in T24 cells, providing new insights into how N-glycosylation drives antitumor drug sensitivity during chemotherapy for patients with cancer.