Hca-F Cells Research Articles

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.

High expression of osteoglycin decreases the metastatic capability of mouse hepatocarcinoma Hca-F cells to lymph nodes

Osteoglycin, one of the matrix molecules, belongs to the small leucine-rich proteoglycan gene family and might play important roles in cell growth and differentiation and in pathological processes such as fibrosis and cancer growth. In this study, a eukaryotic expression plasmid pIRESpuro3 osteoglycin(+) was constructed and transfected into mouse hepatocarcinoma Hca-F cells to evaluate the contribution of osteoglycin to the malignant behavior of Hca-F. It was found that Hca-F cells transfected with pIRESpuro3 osteoglycin(+) showed significantly decreased potential for both migration and invasion. Furthermore, Hca-F cells transfected with osteoglycin showed decreased metastatic potential to peripheral lymph nodes. However, proliferation potential and adhesive capacity of Hca-F cells to different protein substrates were not influenced by osteoglycin transfection. In summary, high expression of osteoglycin decreases the metastatic capability of Hca-F to lymph nodes.

Inhibitory effect of heat shock protein 70 combined with bacillus calmette-guerin on mouse hepatoma Hcaf cells

Inhibitory effect of heat shock protein 70 combined with bacillus calmette-guerin on mouse hepatoma Hcaf cells

RETRACTED: siRNA targeted against matrix metalloproteinase 11 inhibits the metastatic capability of murine hepatocarcinoma cell Hca-F to lymph nodes

Matrix metalloproteinase-11 (MMP-11) belongs to the particular member of MMP family, a group of zinc-dependent endopeptidases involved in tumor progression, invasion and metastasis. MMP-11 is strongly expressed in tumor cells and stromal fibroblasts located in the immediate vicinity of tumor. This study investigated the possible role of MMP-11 expression in mouse hepatocarcinoma cell line Hca-F with highly lymphatic metastasis potential by RNA interference (RNAi) approach. The results showed that a small interfering RNA (siRNA) targeted against MMP-11 significantly impeded Hca-F cells proliferation and colony formation in soft agar, as well as resulted in Hca-F cell apoptosis. This reduction of MMP-11 expression also led to the decreased migration and adhesion of Hca-F cells dramatically both in vitro and in vivo. Furthermore, in vivo metastasis assay indicated that down-regulation of MMP-11 expression in Hca-F cells attenuated the metastatic potential of Hca-F cells to peripheral lymph nodes. These data together provide compelling evidence into the function of MMP-11 and suggest that MMP-11 act as a tumor lymphatic metastasis-associated gene, and could represent a new potential target for gene therapy.

CD147 depletion down-regulates matrix metalloproteinase-11, vascular endothelial growth factor-A expression and the lymphatic metastasis potential of murine hepatocarcinoma Hca-F cells

Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), which is a plasma membrane glycoprotein enriched on the surface of many malignant tumors promotes adhesion, invasion and metastasis of tumor cells. In addition, tumor-associated CD147 also induces vascular endothelial growth factors (VEGFs) expression. To investigate the possible role of CD147 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 CD147 expression. The results showed that CD147 depletion in Hca-F cells resulted in the significantly decreased expression of matrix metalloproteinase-11 (MMP-11), VEGF-A at both mRNA and protein levels. The reduced CD147 expression also attenuated the invasive, adhesive, metastatic ability of Hca-F cells to lymph nodes both in vitro and in vivo. Our current findings reveal that the tumor biological marker CD147 functionally mediates MMP-11, VEGF-A expression and tumor lymphatic metastasis.

Functional expression of CXC chemokine recepter-4 mediates the secretion of matrix metalloproteinases from mouse hepatocarcinoma cell lines with different lymphatic metastasis ability

CXC chemokine recepter-4 (CXCR4) and its ligand, stromal cell-derived factor-1α (SDF-1α) have been implicated in the organ-specific metastasis of several malignancies. Hca-F and its syngeneic cell line Hca-P are mouse hepatocarcinoma cell lines with high and low potential of lymphatic metastasis, respectively. Previous studies showed that the secretion of matrix metalloproteinases (MMPs) associated with the metastatic ability of Hca-F and Hca-P cell line depending on the lymph node environment. However, the mechanism of this process has remained unclear. This study investigated the roles of CXCR4 on Hca-F cell and SDF-1α of lymph node in lymphatic metastasis. The RT-PCR and Flow cytometry analysis results show that Hca-F cells express higher level CXCR4 mRNA and cell-surface CXCR4 protein, as compared with Hca-P cells. Treatment of recombinant SDF-1α proteins induced greater amount of calcium-flux in Hca-F cells than that in Hca-P cells, demonstrating higher functional CXCR4 expression on Hca-F cells than that on Hca-P cells. Furthermore, both the cell-free extratcs of lymph node and recombinant SDF-1α proteins induced secretions of active MMP-9 and MMP-2 from Hca-F cells in vitro. But those secretions were significantly reduced by blockade of cell surface CXCR4 with rabbit anti-mouse CXCR4 polyclonal antibody (pAb) and neutralization of SDF-1α in lymph node extracts with rabbit anti-mouse SDF-1α pAb as well. These results suggest that the CXCR4/SDF-1α system mediates active MMP-9 and MMP-2 secretion from Hca-F and Hca-P cells, which facilitates lymphogenous metastasis of those cells consequently.

Screening and analysis of bioactive compounds in traditional Chinese medicines using cell extract and gas chromatography–mass spectrometry

As the cost of drug development is always many times more than that of drug discovery, predictive methods aiding in the screening of bioavailable drug candidates are of profound significance. In this paper, a novel method for screening bioactive compounds from traditional Chinese medicines (TCMs) was developed by using living cell extract and gas chromatography (GC)–mass spectrometer (MS). The method was validated by using elemene emulsion injection (EEI), a typical TCM with known active compound, to interact with murine ascites hepatocarcinoma cell strain with high metastatic potential (HCa-F). Finally, the method was applied to screen the bioactive compounds from multi-component zedoary turmeric oil and glucose injection (ZTOGI). After HCa-F cells was incubated in ZTOGI, ethyl acetate (EtOAc) was used to extract the compounds in the cells for GC–MS analysis. Fourteen compounds were detected in the desorption eluate of HCa-F cell extract of ZTOGI, and further identified by MS. Curzerene and β-elemene were found to be two major bioactive compounds in ZTOGI. These results show that the method developed may be applied to quickly screen the potential bioactive components in TCMs interacting with the target cells.

Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1–6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1–6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

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 ( approximately 40-60 kDa) and lowly glycosylated form, LG-CD147 ( approximately 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.

Caveolin-1 up-regulates CD147 glycosylation and the invasive capability of murine hepatocarcinoma cell lines

CD147 which is a regulator of matrix metalloproteinase (MMP) production on the surface of many malignant tumor cells, shows a highly specific association with caveolin-1 (Cav-1). As a result of heterogeneous N-glycosylation, CD147 exists in both highly glycosylated form, HG-CD147 (∼40–60 kDa) and lowly glycosylated form, LG-CD147 (∼32 kDa). This study investigated the possible role of Cav-1 in 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, respectively, and in the normal mouse liver cell line IAR-20. Using an RNA interference (RNAi) strategy, we showed that the down-regulation of Cav-1 in Hca-F/RNAi cells could suppress the conversion of LG-CD147 to HG-CD147, down-regulate MMP-11 expression and decrease Hca-F/RNAi cell invasion. Conversely, a stable high expression of Cav-1 in Hepa1-6/Cav-1 cell could cause a specific increase of HG-CD147, up-regulate MMP-11 protein expression and enhance Hepa1-6/Cav-1 cell invasion. In conclusion, Cav-1 expression leads to an increased proportion of HG-CD147 relative to LG-CD147, increased production of MMP-11 and a higher invasive capability. Cav-1 is therefore proposed to act as both an oncogene and a tumor suppressor gene, and could represent a new potential target for gene therapy.

Hyaluronan Promotes Tumor Lymphangiogenesis and Intralymphantic Tumor Growth in Xenografts

Hyaluronan (HA), a high molecular weight glycosaminoglycan in the extracellular matrix, has been implicated in the promotion of malignant phenotypes, including tumor angiogenesis. However, little is known about the effect of HA on tumor-associated lymphangiogenesis. In this study, mouse hepatocellular carcinoma Hca-F cells combined with or without HA were injected subcutaneously into C3H/Hej mice, then angiogenesis and lymphangiogenesis of implanted tumors were examined by immunostaining for platelet-endothelial cell adhesion molecule-1 and lymphatic vascular endothelial hyaluronan receptor-1 respectively. Interestingly, we found HA promotes tumor lymphangiogenesis and the occurrence of intratumoral lymphatic vessels, but has little effect on tumor angiogenesis. Moreover, HA also promotes intralymphatic tumor growth, although it is not sufficient to potentiate lymphatic metastasis. These results suggest that HA, which is elevated in most malignant tumor stroma, may also play a role in tumor progression by promoting lymphangiogenesis.

Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip

In order to obtain lymphogenous metastasis-associated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential. Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip(r) MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics. Out of the 14 500 known genes investigated, 110 (0.8%) were up regulated at least 2(3) fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 2(3) fold. According to the Gene Ontology and TreeView analysis, the 110 genes were further classified into two groups: differential biological process profile and molecular function profile. Using high-throughput gene chip method, a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.

Purification of heat shock protein 70-associated tumor peptides and their antitumor immunity to hepatoma in mice.

To purify the heat shock protein (HSP) 70-associated tumor peptides and to observe its non-MHC-I molecule restrictive antitumor effect. By ConA-sepharose affinity chromatography, ADP-agarose affinity chromatography, and DEAE anion exchange chromatography, we were able to purify HSP70-associated peptides from mouse hepatoma (HCaF) cells treated in heat shock at 42 degrees. Specific active immunization and adoptive cellular immunization assay were adopted to observe the immunoprotective effect elicited by HSP70-associated peptide complexes isolated from HcaF. The finally purified HSP-associated peptides had a very high purity and specificity found by SDS-PAGE and Western blot. Mice immunized with HSP70-associated peptide complexes purified from HCaF cells were protected from HCaF living cell challenge. This effect was dose dependent. Adoptive immunization of immune spleen cells of mice immunized with HSP70-associated peptide complexes could elicit immunity against HCaF challenge, and the tumor-free mice could resist repeated challenges. This effect could be continuously enhanced by repeated challenge with HCaF living cells. The tumor-free mice could tolerate the challenge for as high as 1 x 10(7) HCaF cells. The mice immunized once with spleen cells pulsed with HSP70-associated peptide complexes in vitro could also result in a certain adoptive immunity against HCaF. High purity and specificity of HSP70-associated peptides could be achieved from tumor cells by the low-pressure affinity chromatography method used in this study. HSP70-associated peptide complexes derived from the HCaF can elicit non-MHC-I molecule restrictive immunoprotective effect against HCaF. This effect can be transferred by adoptive immunization to mice and enhanced by repeated challenge with HCaF live cells.

Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential.

In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential. cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5alpha competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing. There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13, ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes. A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.

Different effect of mouse hepatocarcinoma cells with different metastatic potential on host immune system

Objective: To investigate the effect of mouse hepatocarcinoma cell line HCa-F (high metastatic potential) and HCa-P (low metastatic potential) on immune system of the host. Methods: Mice were subcutaneously implanted with HCA-F (F) or HCa-P (P) cells. Cell cycle of lymphocytes from metastatic lymph nodes of tumor-bearing mice was studied by flow cytometry. Fas-L expression of F and P cells was detected immunohistochemically. Apoptosis of macrophages in metastatic lymph nodes was examined by TUNEL methods. Results: For the Hca-F cell bearing mice, the proliferating peak of lymphocytes appeared on 14th day post-inoculation and then decreased rapidly, while for the Hca-P cell bearing mice, the peak appeared on the 7th day post-inoculation and then kept at high level. The expression of Fas-L in F cells was stronger than that in P cells (P<0.01). Apoptosis occurred in macrophages near metastatic F cells in lymph nodes of tumor-bearing mice. Conclusion: Carcinoma cells with high metastatic potential may suppress immune reaction of host through inducing apoptosis of macrophages (one of the important antigen present cells) by Fas-L-Fas interreaction. And the expression of Fas-L in tumor cells may be associated with high metastatic ability to lymph nodes.

Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice.

To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P<0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P <0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression of Fas ligand protein of Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.