Shujing Wang

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.

Deglycosylation of CD147 down‐regulates Matrix Metalloproteinase‐11 expression and the adhesive capability of Murine hepatocarcinoma cell HcaF in vitro

CD147 is a plasma membrane glycoprotein, enriched on the surface of many malignant tumor cells. As a result of heterogeneous N‐glycosylation, CD147 exists in both a highly glycosylated form, HG‐CD147 (∼40 ‐ 60kDa) and lowly glycosylated form, LG‐CD147 (∼32 kDa). This experiment investigated the possible role of CD147 glycosylation in the HcaF, HcaP and Hepa1‐6 mouse hepatocarcinoma cell lines, which have high, low and no metastatic potential in the lymph nodes. Western blot analysis showed that the ratio of HG‐CD147/LG‐CD147 protein expression on HcaF and HcaP were much higher than that on Hepa1‐6 cells. By treatment with tunicamycin (TM), an inhibitor of N‐glycosylation, the expression level of HG‐CD147 decreased and the LG‐CD147 disappeared completely in HcaF cells. Meanwhile, Matrixmetallproteinase‐11 (MMP‐11) protein expression was down‐regulated, and the adhesive capability of HcaF cells to endothelial cells in cryosection of mouse lymph nodes decreased. These results indicated that the glycosylation of CD147 plays a crucial role. It is HG‐CD147 that may contribute more to tumor progress, invasion and metastasis into lymph node rather than LG‐CD147. The results of this study are of biological and clinical importance. iubmb Life, 58: 209‐216, 2006

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.

α2,6-linked sialic acids on N-glycans modulate the adhesion of hepatocarcinoma cells to lymph nodes

The alterations of cell surface sialylation play a key role in tumor metastasis. Enhanced α2,6-sialylation on N-glycans results from overexpression of the sialyltransferase ST6Gal-I. Hca-F and Hca-P cells are murine hepatocarcinoma cell lines which metastasize only to the lymph nodes. Our previous study revealed that ST6Gal-I was involved in the adhesion of Hca-F cells to fibronectin. However, the roles of sialic acids in the adhesion of Hca-F cells to lymph nodes still remain poorly understood. In this study, we observed that expression levels of α2,6-linked sialic acids on Hca-F cells were higher compared to Hca-P cells. Knockdown of ST6Gal-I by small hairpin RNA (shRNA) transfection decreased the expression of α2,6-linked sialic acids and inhibited the adhesion of Hca-F cells to lymph nodes. The adhesion ability was reported to be mediated by siglec-2 which preferentially binds to α2,6-linked sialic acids, and in addition, ST6Gal-I knockdown inhibited the phosphorylated levels of focal adhesion kinase (FAK) and paxillin when cells were treated with siglec-2. Taken together, these results suggest that interaction of α2,6-linked sialic acids with siglec-2 might modulate the adhesion of hepatocarcinoma cells to lymph nodes through the FAK signaling pathway.

ST6Gal-I modulates docetaxel sensitivity in human hepatocarcinoma cells via the p38 MAPK/caspase pathway

The β-galactoside α2-6-sialyltransferase 1 (ST6Gal-I) is the principal sialyltransferase responsible for the addition of α2-6-sialic acid to the termini N-glycans on cell surface. Although ST6Gal-I in cancer cell resistance to chemotherapeutics agents has been previously reported, the role of ST6Gal-I in clinical drug resistance of hepatocellular carcinoma (HCC) is not fully understood. In this study, we found that knockdown of ST6Gal-I increased the sensitivity of hepatocarcinoma MHCC97-H cells to docetaxel treatment by instigating the process of apoptosis. Silencing ST6Gal-I expression decreased the survival rate of MHCC97-H cells after docetaxel treatment. Importantly, ST6Gal-I silencing resulted in an increasing of phospho-p38, Bax, Bad, cytochrome c and the cleaved caspase-9, 3 and PARP, while a decreasing of the anti-apoptotic protein Bcl-2. In addition, we found that p38 MAPK and caspase-3 inhibitors can reduce the enhanced apoptosis levels of MHCC97-H cells resulted by either ST6Gal-I silencing or docetaxel treatment. Conversely, exogenous expression of ST6Gal-I in hepatocarcinoma Huh7 cells inhibited apoptotic cell death and prevented docetaxel-induced apoptosis by inhibiting p38 MAPK mediated mitochondrial-dependent pathway. Taken together, these results indicate that ST6Gal-I might play a positive role in mediating the survival of human hepatocarcinoma cells and could be a potential target for gene and antitumor drugs therapy.

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.

Caveolin‐1 promotes the transformation and anti‐apoptotic ability of mouse hepatoma cells

Caveolin‐1 is a major structural protein of caveolae and plays important roles in signal transduction, cellular transformation and tumor metastasis. Our previous study demonstrated that caveolin‐1 expression level was positively correlated with the invasive ability of mouse hepatoma Hepa1‐6 and Hca‐F cells. However, the role of caveolin‐1 in cellular transformation and apoptosis remains undetermined. We found that exogenous expression of caveolin‐1 in Hepa1‐6 cells enhanced cell transformation capability both in vitro and in vivo and prevented actinomycin D‐induced apoptosis via the activation of survivin‐mediated survival pathway. Conversely, downregulation of caveolin‐1 in Hca‐F cells significantly attenuated cell transformation ability in vitro and in vivo and increased cell sensitivity to actinomycin D by inhibiting survivin‐mediated survival pathway. These results indicate that caveolin‐1 could play an active role in mediating the transformation and survival of mouse hepatoma cells and might be a potential target for gene and antitumor drugs therapy.

Livin regulates prostate cancer cell invasion by impacting the NF-κB signaling pathway and the expression of FN and CXCR4

Prostate cancer (PCa) has the second highest mortality rate of all tumor‐related diseases for males in Western countries, and the incidence of PCa in China is increasing. Previous studies have proven that inhibitor of apoptosis proteins (IAPs) can regulate tumor cell invasion and metastasis. Livin is the most recently identified IAP. Our previous study showed that Livin might play an important role in the initiation of human PCa and that Livin‐α might promote cell proliferation by regulating the G1–S cell cycle transition. However, whether Livin, as an IAP, can regulate the invasive ability of PCa cells remains unknown. In this study, we found that the expression of Livin was higher in metastatic PCa tissues than in nonmetastatic tissues and that the expression of Livin was downregulated/upregulated by small interfering RNA/vector, which could inhibit/promote PC‐3/LNCaP cell invasion. This action was related to the impact of Livin on nuclear factor‐κB (NF‐κB) and its downstream signaling pathway, including FN and CXCR4. Together, our findings suggested that Livin might regulate tumor cell invasion in PCa directly, and that Livin might be an ideal candidate for preventing tumor cell invasion.