Yongfu Zhao

Modification of Sialylation Mediates the Invasive Properties and Chemosensitivity of Human Hepatocellular Carcinoma

Aberrant sialylation is closely associated with malignant phenotypes of tumor cells, including invasiveness and metastasis. This study investigated sialylation with regard to the modification of invasive properties and chemosensitivity in human hepatocellular carcinoma (HCC) cell lines and the association between the sialyltransferase gene family and clinicopathological characteristics in HCC patients. Using mass spectrometry analysis, we found that the composition profiling of sialylated N-glycans differed between MHCC97H and MHCC97L cells with different metastatic potential. The expressional profiles of 20 sialyltransferase genes showed differential expression in two cell lines, transitional and tumor tissues, from the same patients. Two genes, ST6GAL1 and ST8SIA2, were detected as overexpressed in MHCC97H and MHCC97L cells. The altered expression levels of ST6GAL1 and ST8SIA2 corresponded to a changed invasive phenotype and chemosensitivity of MHCC97H and MHCC97L cells both in vitro and in vivo. Further data indicated that manipulation of the expression of the two genes led to altered activity of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway. Targeting the PI3K/Akt pathway by its specific inhibitor wortmannin or by Akt RNA interference resulted in a reduced capacity for invasion and chemoresistance of MHCC97H cells. Our results imply that sialylation may function as an internal factor, regulating the invasion and chemosensitivity of HCC, probably through ST6GAL1 or ST8SIA2 regulation of the activity of the PI3K/Akt pathway.

Differential expression of Axl in hepatocellular carcinoma and correlation with tumor lymphatic metastasis

Protein kinases play important roles in tumor development and progression. A variety of members of the signal transduction enzymes serve as targets for therapeutic intervention in cancer. The dysregulation of Axl receptor and its ligand growth arrest‐specific 6 (Gas6) is implicated in the pathogenesis of several cancers. In this study, the differential expressions of Axl were investigated in mouse hepatocarcinoma cell lines Hca‐F and Hca‐P, which have high‐ and low‐metastatic potential to lymph nodes. Experimental inhibition of Axl by siRNA assessed further the metastatic potential of Axl. The results showed that down‐regulation of Axl expression attenuated Hca‐F cells proliferation, migration, and invasion in vitro, as well as inhibited metastasis to peripheral lymph nodes in vivo. Further analysis demonstrated that the addition of exogenous Gas6 mediated the migration and invasion of Hca‐F cells both in vitro and in vivo through Axl. Furthermore, Gas6 stimulation of Axl in Hca‐F cells resulted primarily in the down‐regulation of Cyr61, a member of the CCN protein family involved in tumor progression. These data suggest that Axl acts as a tumor lymphatic metastasis‐associated gene, and may function partly through the regulation of Cyr61.

Glycomic alterations are associated with multidrug resistance in human leukemia.

Correlations of disease phenotypes with glycosylation changes have been analyzed intensively in tumor biology field. In this study we describe glycomic alterations of multidrug resistance in human leukemia cell lines. Using multiple glycan profiling tools: real-time PCR for quantification of glycogenes, FITC-lectin binding for glycan profiling, and mass spectrometry for glycan composition, we compared the glycomics of drug-resistant K562/ADR cells with parental K562 line. The results showed that the expression of glycogenes, glycan profiling and N-glycan composition were different in K562/ADR cells, as compared with those in K562 cells, whereas O-glycans of the two cell lines showed no different mass spectra. Further analysis of the N-glycan regulation by way of tunicamycin application or PNGase F treatment in K562/ADR cells showed partial inhibition of biosynthesis and increased sensitivity to chemotherapeutic drugs in vitro. We targeted glycogene B3GNT8 and ST8SIA4, which were over-expressed in K562/ADR cells, and silenced the expression levels of two glycogenes after using RNA interference approach. The results showed that the silencing of B3GNT8 or ST8SIA4 in K562/ADR cells resulted in increased chemosensitivity to anti-tumor drugs. In conclusion, glycomic alterations are responsible for the overcoming multidrug resistance in human leukemia therapy and the N-linked oligosaccharides are associated with the drug resistance of cancer cells.

Axl gene knockdown inhibits the metastasis properties of hepatocellular carcinoma via PI3K/Akt-PAK1 signal pathway.

The objective of this study is to clarify the possible role and mechanism of Axl in the tumorigenicity and metastasis process of hepatocellular carcinoma. The mRNA and protein expression levels of Axl in MHCC97-H and MHCC97-L cell lines were evaluated by real-time PCR and Western blot analysis. The key factor of phosphatidylinositol-3-kinase (PI3K)/Akt-p21-activated kinases-1 (PAK1) signaling pathway was studied after Axl expression was downregulated by shRNA. Finally, we analyzed the expression status of Axl protein expression in hepatocellular carcinoma tissues and its relationship with the prognosis of hepatocellular carcinoma. Axl was observed to be higher expressed in MHCC97-H cell lines compared to MHCC97-L cell lines. The downregulation of Axl in MHCC97-H cell lines resulted in the inhibition of the invasion ability of MHCC97-H cells both in vitro and in vivo. Interestingly, blocking PI3K/Akt signaling pathway by LY294002 or Akt siRNA could remarkably inhibit the PAK1 activation and cell invasion. Finally, the Axl protein expression was positively correlated with differentiation, lymph node metastasis, and clinical stage in patients with hepatocellular carcinoma patients (all P < 0.01). These findings suggest that Axl can also regulate the metastasis process of hepatocellular carcinoma and may serve as a new prognostic marker and therapeutic target for treating hepatocellular carcinoma metastasis.

Axl mediates tumor invasion and chemosensitivity through PI3K/Akt signaling pathway and is transcriptionally regulated by slug in breast carcinoma.

The invasion and chemoresistance are crucial causes of morbidity and relapse for cancer patients. Axl is implicated in the modulation of cell invasion, cancer metastasis, and chemosensitivity in human breast carcinoma cell lines. Both breast cancer cell lines and tissues displayed increased expression of Axl, and it over expressed in highly metastatic breast cancer. The altered expression level of Axl was corresponding to the changed invasive phenotype and chemosensitivity of MDA‐MB‐231 cells both in vitro and in vivo. Further data indicated that experimental inhibition of Axl by RNAi assay inhibited phosphatidylinositol 3‐kinase (PI3K)/Akt/GSK3β signaling pathway, resulted in the decrease of Slug expression, and further suppressed cell invasion properties and chemosensitivity. What is more, after the detection and statistics in human breast cancer specimens, we found the Axl expression was closely correlated with histological grade, lymph node metastasis, and clinical stage (P < 0.01). Taken together, these findings indicate that Axl exerts the role of tumor metastasis and chemosensitivity through activation of the PI3K/Akt/GSK3β signaling pathway, which is transcriptionally regulated by Slug. Our findings support the possibility that Axl is a novel regulator. It means by targeting Axl or its related signaling pathways, we can reduce the invasion and chemosensitivity of breast tumor.

Expression of CD147 mediates tumor cells invasion and multidrug resistance in hepatocellular carcinoma.

Multidrug resistant (MDR) tumor cells over-expressing P-glycoprotein exhibit variation in invasive behavior. To investigate the mechanisms, we analyzed the expression of CD147. The results showed that CD147 expression was increased in HepG2/Adr cells, as compared to HepG2 cells. The MDR cells produced more MMP11 and MDR1, which promoted HepG2/Adr cells invasion and increased resistance to chemotherapeutic drugs. On the other hand, CD147 silencing in HepG2/Adr cells by RNAi led to the opposite effect. Treatment of tumor cells with U-0126, an inhibitor of MAPK/Erk, also down-regulated MMP11 and MDR1 expression. Thus, CD147 may functionally mediate tumor cells invasion and MDR.

Differential Expression of Axl and Correlation with Invasion and Multidrug Resistance in Cancer Cells

Dysregulation of Axl receptor tyrosine kinases is implicated in the pathogenesis of several sensitive and refractory cancers. In this study we showed that Axl expression was upregulated in drug-resistant cancer cells, as compared to those in parental cells. Downregulation of Axl expression by RNAi led to a decrease of K562/ADR and MCF-7/ADR cells invasion, proliferation in soft agar, and increased cells chemosensitivity in vitro. In nude mice bearing xenografts, downregulation of Axl in MDR cells enhanced the anticancer activity of intraperitoneally applied chemotherapeutic drugs. Our observations suggest that Axl represents a promising gene for overcoming MDR in cancer therapy.

siRNA‐targeted COL8A1 inhibits proliferation, reduces invasion and enhances sensitivity to D‐limonence treatment in hepatocarcinoma cells

The COL8A1 (collagen type VIII, alpha‐1) gene, which encodes the alpha 1 chain of collagen, type VIII, may modulate migration, proliferation and adherence of various cells. Only very sparse information exists on COL8A1 expression in hepatocarcinoma. To investigate the possible role of COL8A1 in the mouse hepatocarcinoma cell line Hca‐F with highly metastatic potential in the lymph nodes, we used an RNA interference (RNAi) approach to silence COL8A1 expression. The results showed that a small interfering RNA (siRNA) targeted against COL8A1 significantly impeded Hca‐F cells proliferation and colony formation in soft agar. This reduction of COL8A1 expression also led to the decreased invasion of Hca‐F cells dramatically in vitro. Furthermore, the downregulation of COL8A1 expression also sensitized cells to the action of D‐limonene. These data together provide insights into the function of COL8A1 and suggest that COL8A1 might represent a new potential target for gene therapy in hepatocarcinoma.

Axl as a downstream effector of TGF-β1 via PI3K/Akt-PAK1 signaling pathway promotes tumor invasion and chemoresistance in breast carcinoma

The invasion and chemoresistance are crucial causes of morbidity and death for cancer patients. Axl is closely associated with malignant phenotype of breast tumor cells, including invasiveness and metastasis. Both breast cancer cell line and tissue displayed increased expression of Axl, especially in highly metastatic breast cancer. On the contrary, experimental inhibition of Axl or transforming growth factor beta 1 (TGF-β1) by RNAi assay could suppress cell invasion ability and chemoresistance. Moreover, the up-regulation of Axl was induced by TGF-β1, further activated phosphatidylinositol 3-kinase (PI3K)/Akt and PAK1 translocation, and resulted in greater cell motility, invasion, and chemoresistance in vitro and in vivo. After the detection and statistics in human breast cancer specimens, we found that the Axl expression was closely correlated with TGF-β1 level, tumor differentiation, lymph node metastasis, and clinical stage (p < 0.01). Our findings support the possibility that Axl is a significant regulator of invasion and chemosensitivity, and it means by targeting Axl or its related signaling pathways, we can reduce the invasion and chemosensitivity of breast tumor.

Glycogenes mediate the invasive properties and chemosensitivity of human hepatocarcinoma cells.

Aberrant cell-surface glycosylation patterns are present on tumors and have been linked to tumor progression. This study aimed to identify the alterations of glycogene and N-glycan involved in tumor invasion, tumorigenicity and drug resistance in MHCC97-H and MHCC97-L human hepatocarcinoma cell lines, which have high, low metastatic potential, respectively. Using real-time PCR for quantification of glycogene and FITC-lectin binding for glycan profiling, we found that the expression of glycogenes and glycan profiling were different in MHCC97-H cells, as compared to those in MHCC97-L cells. We silenced the expression levels of glycogenes MGAT3 and MGAT5, which were over-expressed in MHCC97-L and MHCC97-H cells. Knockdown of MGAT3 expression promoted MHCC97-L cells invasion and increased resistance to 5-fluorouracil in vitro. The silencing of MGAT5 in MHCC97-H cells inhibited invasion and increased sensitivity to 5-fluorouracil in vitro. Further analysis of the N-glycan regulation by tunicamycin application or PNGase F treatment in MHCC97-H and MHCC97-L cells showed partial inhibition of N-glycan glycosylation, decreased invasion, tumorigenicity and increased sensitivity to 5-fluorouracil both in vitro and in vivo. These findings suggest that alterations of glycogene and N-glycan in human hepatocarcinoma cells correlate with tumor invasion, tumorigenicity and sensitivity to chemotherapeutic drug, and have significant implications for the development of treatment strategies.