Alreration of nuclear matrix-intermediate filament system and differential expression of nuclear matrix proteins during human hepatocarcinoma cell differentiation

AIM： To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. METHODS： Cells cultured with or without 5 × 10^-3 mmol/L of hexamethylene bisacetamide （HMBA） on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser- desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Data were submitted for database searching using Mascot tool （www.matrixscience.com）. RESULTS： The nuclear matrix （NM） and intermediate filament （IF） in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched utrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate- sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pystl, hypothetical protein, nucleophosminl, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, 13-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. CONCLUSION： The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament （NM-IF） system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion.

Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays

AIM： To demonstrate the feasibility of using woodchuck samples on human microarrays, to provide insight into pathways involving positron emission tomography （PET） imaging tracers and to identify genes that could be potential molecular imaging targets for woodchuck hepatocellular carcinoma. METHODS： Labeled cRNA from woodchuck tissue samples were hybridized to Affymetrix U133 plus 2.0 GeneChips. Ten genes were selected for validation using quantitative RT-PCR and literature review was made. RESULTS： Testis enhanced gene transcript （BAX Inhibitor 1）, alpha-fetoprotein, isocitrate dehydrogenase 3 （NAD＋） beta, acetyI-CoA synthetase 2, carnitine palmitoyltransferase 2, and N-myc2 were up-regulated and spermidine/spermine N1-acetyltransferase was down-regulated in the woodchuck HCC. We also found previously published results supporting 8 of the 10 most up-regulated genes and all 10 of the 10 most downregulated genes. CONCLUSION： Many of our microarray results were validated using RT-PCR or literature search. Hence, we believe that woodchuck HCC and non-cancerous liver samples can be used on human microarrays to yield meaningful results.

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

AIM： To construct an expression plasmid encoding human wild-type midkine （MK） and enhanced green fluorescence protein （EGFP） fusion protein （MK-EGFP）, and to analyze the subcellular localization of MK in different cardnoma cell lines. METHODS： Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS： Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION： This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

Expressions of Survivin and Caspase-3 in human hepatocellular carcinoma and the relationship with prognosis

Objective: The aim of our study was to investigate the expressions of Survivin and Caspase-3 in the hepatocellular carcinoma (HCC) and the paracancerous liver tissues, and define the relationship between the expression and the patients survival rate after surgery. Methods: The expressions of PCNA, Survivin and Caspase-3 were detected by immunohistochemistry. PCNA positive level was used to account proliferating cell nuclear antigen proliferating index (PCNA-PI). The apoptosis index of HCC tissues was detected by TUNEL. Results: The expression of Survivin in HCC tissue was higher than that in paracancerous tissue (P < 0.05), while it was related with portal vein thrombosis, tumor size and histological differentia- tion (P < 0.05). In HCC tissues, the PCNA-PI of the Survivin positive group was higher than the negative (P < 0.05) group, and the AI of the Survivin positive group was lower than the negative group (P < 0.05). The expression of Caspasse-3 in HCC tissue was lower than that in paracancerous tissue (P < 0.05), while it was related with HBsAg and histological differentiation (P < 0.05). In HCC tissues, the average PCNA-PI of the Caspase-3 positive group was lower than that of the negative group (P < 0.05), and the average AI of the Caspase-3 positive group was higher than that of the negative group (P < 0.05). There was negative correlation between the expressions of Survivin and Caspase-3 in HCC (r = -0.242, P < 0.05). The survival rate of the Survivin positive group was lower than that of the negative group (P < 0.05), while that of the Caspase-3 positive patients was higher than that of the negative group (P < 0.05). Conclusion: The expressions of Survivin and Caspase-3 in HCC can reflex the biological behavior of HCC and patients prognosis, they may be new targets of tumor diagnosis and treatment.

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53, proliferating cell nuclear antigen (PCNA) and apoptosis in patients with hepatocellular carcinoma (HCC) after surgical tumor ablation. METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry. Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick-end labeling (TUNEL) method was used to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0 statistical package. RESULTS: TAp73α overexpressed in HCC tissues (36.2%) when compared with adjacent non-cancerous tissues (2.38%, P<0.005) and normal liver tissues (0, P<0.01). Mutant type p53 (mt-p53) overexpressed in HCC tissues (38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P<0.05) and normal liver tissues (0, P<0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%±4.46% vs 27.88%±5.89%, t, P=0.028). Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%±2.28% vs 7.38%±2.61%, t, P=0.019). Expression of TAp73a was associated with lymph node metastasis and mt-p53, with r=0.407 and 0.265, respectively. Expression of mt-p53 was associated with Edmondson's stage and AFP, with r=0.295 and-0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P=0.039, 0.012, 0.002, 0.000, 0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and age were independent factors of prognosis. CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.

β-catenin accumulation in nuclei of hepatocellular carcinoma cells up-regulates glutathione-s-transferase M3 mRNA

AIM: To identify the differentially over-expressed genes associated with β-catenin accumulation in nuclei of hepatocellular carcinoma (HCC) cells. METHODS: Differentially expressed genes were identified in radiation-induced B6C3 F1 mouse HCC cells by mRNA differential display, Northern blot and RT-PCR, respectively. Total glutathione-s-transferase (GST) activity was measured by GST activity assay and β-catenin localization was detected with immunostaining in radiation-induced mouse HCC cells and in HepG2 cell lines.RESULTS: Two up-regulated genes, glutamine synthetase and glutathione-s-transferase M3 (GSTM3), were identified in radiation-induced mouse HCC cells. Influence of β-catenin accumulation in nuclei of HCC cells on upregulation of GSTM3 mRNA was investigated. The nearby upstream domain of GSTM3 contained the β-catenin/TcfLef consensus binding site sequences [5'-(A/T)(A/T) CAAAG-3'], and the total GST activity ratio was considerably higher in B6C3F1 mouse HCC cells with β-catenin accumulation in nuclei of HCC cells than in those without β-catenin accumulation (0.353 ± 0.117vs 0.071 ± 0.064, P < 0.001). The TWS119 (a distinct GSK-3β inhibitor)-induced total GST activity was significantly higher in HepG2 cells with β-catenin accumulation than in those without β-catenin accumulation in nuclei of HCC cells. Additionally, the GSTM3 mRNA level was significantly higher at 24 h than at 12 h in TWS119-treated HepG2 cells. CONCLUSION: β-catenin accumulation increases GST activity in nuclei of HCC cells, and GSTM3 may be a novel target gene of the β-catenin/Tcf-Lef complex.

In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles

AIM:To synthesize antisense oligonucleotides (ASODNs) of midkine (MK), package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma (HCC) growth using these nanoparticles.METHODS: HepG2 cell proliferation was analyzed in vitro using the 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction (PCR). RESULTS: The in vitro proliferation of HepG2 cells was signif icantly inhibited by the nanoparticles packaged with MK-ASODNs (NANO-ASODNs). Furthermore, the NANO- ASODNs signif icantly inhibited the growth of HCC in the mouse model. CONCLUSION: NANO-ASODNs can significantly suppress the growth of HCC in vitro and in vivo.

Apoptosis induced by nucleosides in the human hepatoma HepG2

AIM： To investigate the apoptotic effects of nucleosides on the human hepatoma HepG2. METHODS： The nucleosides included inosine （I）, cytidine （C）, uridine （U）, thymidine （T）, adenosine （A）, and guanosine （G）. Cells were incubated by the mediums with or without nudeosides at 37 ℃ in a 50 mL/L CO2 humidified atmosphere. RESULTS： It was found that the cell viabilities were significantly decreased, when cells were treated with 30 mmol/L I, 30 mmol/L C, 30 mmol/L U, 30 mmol/L T, 0.5 mmol/L A, and 0.5 mmol/L G after 12 h incubation （P〈0.05）. About the apoptotic phenomenon, the cell percentages of sub-G1 cells were significantly increased in the mediums containing nucleosides such as C, U, T, A, and G （P〈0.05）. Furthermore, the caspase-3 activity was increased, when the cells were incubated with T （P〈0.05）. The protein expressions of p53 and p21 showed no difference in each group. To investigate the mechanism of apoptosis induced by nucleosides, it was found that the contents of soluble Fas ligand contents were increased in HepG2 cells following I, U, T, and A treatment （P〈0.05）. But, TNF-α and cytochrome cwere undetectable. CONCLUSION： Thymidine may induce the apoptosis in HepG2, but the effective dosages and reactive time must be investigated in the future study. However, the apoptosis-inducing abilities of other nucleosides were still unclear in this study.