Wan-Long Chuang

Genes responsible for the characteristics of primary cultured invasive phenotype hepatocellular carcinoma cells

The common genes responsible for the characteristics of primary cultured invasive phenotype hepatocellular carcinoma (HCC) cells were investigated. Primary cultured HCC cells from three patients were separated by Matrigel invasion into parent and invasive cells. Whole human genome oligo microarray was applied to detect the differentially expressed genes in invasive cells. A purchased HCC cell line (HA 22T/VGH) was studied for comparison. Forty genes were consistently up-regulated and 14 genes were consistently down-regulated among primary cultured invasive cells. Among these genes, only three up-regulated genes (CNN1, PLAT, SPARC) and one down-regulated tumor suppressor gene (MDFI) had same expressions in invasive cells originated from purchased cell line. For primary cultured invasive cells, differential expressions of several groups of common genes are known to have capacities to promote proliferation (CAV1, IL6, PLAT, RRAD, SRPX), remodeling of extracellular matrix (COL1A1, COL1A2, NID2, TNC, RELN, SPARC), migration (ACTG2, CAV1, CCL2, CCL26, CDC42EP3, CNN1, PHLDB2, PLAT, RRAD, SRPX), implantation (IL6), immune escape (CD70) and angiogenesis (CCL2, IL6, IL18, PLAT, SLIT3). Two genes related to signal transduction (AXL, RASL10B) and one related to metabolism (PTGS2) also showed consistent expressions. Differential expressions of these genes are capable for tumor invasiveness. In conclusion, the characteristics of invasive phenotype HCC cells are originated from differential expressions of several groups of genes rather than few target genes. This information may give us a new insight to design new stratagems in HCC treatment. Analysis of the results from a purchased cell line may have bias due to long-term repeated in vitro cultures.

Cancer-associated fibroblasts up-regulate CCL2, CCL26, IL6 and LOXL2 genes related to promotion of cancer progression in hepatocellular carcinoma cells.

Impact of different cancer-associated fibroblast (CAF) cell lines on proliferation, migration, invasion and differential expressions of genes in different hepatocellular carcinoma (HCC) cell lines was investigated. Two human CAF cell lines (F26/KMUH, F28/KMUH) and two human HCC cell lines (HCC24/KMUH, HCC38/KMUH) were studied. Influence of F28/KMUH cells on expressions of genes in HCC38/KMUH cells was detected by microarray to select genes for further analysis. Both CAF cell lines promoted proliferation (all P<0.05), migration (all P<0.05) and Matrigel invasion (all P<0.0001) of both HCC cell lines. F26/KMUH cells showed stronger promoted effects on, firstly, proliferation of HCC24/KMUH cells (P=0.0064) and, secondly, migration of both HCC cell lines than F28/KMUH cells did (all P<0.002). Ten up-regulated genes (APLN, CCL2, CCL26, CXCR4, IL6, MUC1, LOXL2, PDGFA, PGK1, VEGFA) related to proliferation, migration, invasion and angiogenesis of HCC detected by microarray were selected for quantitative reverse transcriptase-polymerase chain reaction analysis. Both CAF cell lines had same tendency of effects on differential expressions of genes in same HCC cell line, but expressions of genes between different HCC cell lines were not consistent. Only CCL2, CCL26, IL6 and LOXL2 genes were consistently up-regulated in both HCC cell lines. In conclusion, the effects of CAFs to promote proliferation, migration and invasion of HCC cells are influenced by the characteristics of both CAFs and HCC cells. Up-regulations of CCL2, CCL26, IL6 and LOXL2 genes in cancer cells are part of the common effects of CAFs on HCC cells.

Anti-cancer mechanisms of clinically acceptable colchicine concentrations on hepatocellular carcinoma

AIMS This study was to investigate whether the clinically acceptable colchicine concentrations had anti-cancer effects on hepatocellular carcinoma (HCC) and their anti-cancer mechanisms. MAIN METHODS Two human HCC cell lines (HCC24/KMUH, HCC38/KMUH) and two human cancer-associated fibroblast (CAF) cell lines (F28/KMUH, F59/KMUH) were investigated by proliferative assay, microarray, quantitative reverse transcriptase-polymerase chain reaction, and nude mouse study using clinically acceptable colchicine concentrations. KEY FINDINGS Both 2 and 6ng/mL colchicine significantly inhibited the cellular proliferation of all cell lines tested (P<0.05). The anti-proliferative effects of colchicine on F28/KMUH, HCC24/KMUH and HCC38/KMUH cells were dose-dependent. The anti-proliferative effects of 6ng/mL colchicine on both HCC cell lines were similar to the effects of 1μg/mL epirubicin. The anti-proliferative effects of colchicine on HCC cells could be partially explained by dose-dependent up-regulations of 2 anti-proliferative genes (AKAP12, TGFB2) in these cells. TGFB2 was also up-regulated in CAFs but was not dose-dependent. Up-regulation of MX1 which can accelerate cell death was a common effect of 6ng/mL colchicine on both CAF cell lines, but 2ng/mL colchicine down-regulated MX1 in F28/KMUH cells. Nude mouse (BALB/c-nu) experiment showed that colchicine-treated mice (0.07mgcolchicine/kg/day×14days) had lower increased tumor volume ratios, slower tumor growth rates and larger percentages of tumor necrotic areas than control mice (all P<0.05). SIGNIFICANCE Clinically acceptable colchicine concentrations have anti-cancer effects on HCC. This drug has potential for the palliative treatment of HCC.

Amphotericin B up-regulates angiogenic genes in hepatocellular carcinoma cell lines.

Background  Amphotericin B (AmB) has a discordant influence on epirubicin (4′‐epidoxorubicin) cytotoxicity in hepatocellular carcinoma (HCC). This indicates that the cellular function of HCC may be significantly influenced by AmB. Whether the influence of AmB on HCC has any possibility to influence cancer growth has not been studied. This study was to try and clarify this issue.

Myocyte apoptosis in the pathogenesis of ureteral damage in rats with obstructive uropathy.

OBJECTIVES To elucidate the role of signal apoptosis in the pathogenesis of ureteral damage during the course of obstructive uropathy and to investigate the cell proliferation in the smooth muscle layer of ligated ureter. METHODS The apoptotic cells were detected with the method of in situ end-labeling of DNA fragments. The expression of Fas, tumor necrosis factor receptor 1 (TNF-R1), and proliferation cell nuclear antigen (PCNA) was examined in 54 rats by immunohistochemistry. RESULTS The severity of hydroureter and the histologic changes of ureteral smooth muscle were aggravated during the period of obstruction. The apoptotic cells and the expression of Fas and PCNA in the smooth muscle layer were present since day 14 after ligation. The percentages of apoptotic cells and the expression indexes of Fas and PCNA in the smooth muscle layer progressively increased, reaching a peak on day 21 after ligation, and then declined. The expression of TNF-R1 in the smooth muscle layer was only found on day 21 after ligation. The numbers of the apoptotic cells in the smooth muscle layer correlated significantly with the expression of PCNA, Fas, and TNF-R1. The expression of Fas and TNF-R1 in the smooth muscle layer also correlated significantly. The appearance of apoptotic cells and the expression of Fas and PCNA in the smooth muscle layer were associated with tissue damage and fibrosis in the smooth muscle layer. CONCLUSIONS We conclude that cell apoptosis and the expression of Fas, TNF-R1, and PCNA might play important roles in the pathogenesis of ureteral damage in the smooth muscle layer of obstructed ureters.

Inhibition of nuclear factor-kappa B (NF-κB) activation attenuates ureteric damage in obstructive uropathy

OBJECTIVE Nuclear factor (NF)-kappaB is a ubiquitous transcription factor that can be activated by multiple signals. To elucidate the role of NF-KB and the effects of NF-kappaB inhibitor on ureteric damages in obstructive uropathy, we conducted this study. METHODS The expressions of NF-kappaBp50, NF-kappaBp65, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), proliferation cell nuclear antigen (PCNA) and cell apoptosis were examined in 80 rats. Pyrrolidine dithiocarbamate (PDTC) was administered to 40 rats. The others served as controls. RESULTS After ureteric ligation, hydroureter and ureteric damages progressively aggravated. But the severity of hydroureter and fibrosis of muscle layer in the ligated ureters in the treated group were significantly milder than those of the control group. The expressions of NF-kappaBp50 and NFkappa-Bp65 in the smooth muscle layer of obstructed ureters were found in the rats in control group from the day 14 after ureteric ligation. The expressions of NF-kappaBp65 and NF-kappaBp50 in the nuclei of muscle cells in obstructed ureters were correlated significantly with the expressions of IL-6, TNF-alpha, PCNA and the number of the apoptotic cells. The expressions of NF-kappaBp50, NF-kappaBp65 in the nuclei of myocytes and fibrotic changes of smooth muscle layer were correlated significantly. Treatment with PDTC diminished the expressions of NF-kappaBp50 and NF-kappaBp65. The expression of IL-6, TNF-alpha, PCNA and the labeling index of apoptotic cells in the smooth muscle layer of ligated ureters in the PDTC-treated group were also decreased. CONCLUSIONS We concluded that expression of NF-kappaB might contribute to the ureteric damage in obstructive uropathy, and that inhibition of NF-kB could attenuate the tissue damages of obstructed ureters.

Atorvastatin ameliorates tissue damage of obstructed ureter in rats

AIMS To investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor on the tissue damage and fibrosis of obstructed ureters, 80 rats were studied. MAIN METHODS Atorvastatin, a HMG-CoA reductase inhibitor, was administered to 40 rats at the dose of 20 mg/kg per day 1day before unilateral ligation of ureters and every day thereafter. The other rats served as controls. Eight rats from each group were sacrificed for examination on days 7, 14, 21, 28 and 42 after ligation, respectively. The expressions of transforming growth factor-β1 (TGF-β1), Interleukine-1β (IL-1β), Interleukine-6 (IL-6), tumor necrosis factor-alpha (TNF-α), proliferation cell nuclear antigen (PCNA), and the apoptotic cells in the ureteric smooth muscle were examined. KEY FINDINGS Hydroureter and fibrosis of the muscle layer became progressively aggravated in the ligated ureters of the atorvastatin-treated group and control group. The severities of hydroureter and muscle layer fibrosis in the ligated ureters of the treated group were significantly less than in the control group. The atorvastatin administration also decreased the expression of TGF-β1, IL-1β, IL-6, TNF-α, PCNA and the labeling index of apoptotic cells in the smooth muscle layer of ligated ureters in the treated group. SIGNIFICANCE We concluded that atorvastatin might ameliorate the tissue damage of obstructed ureters, at least partially, via the inhibition on TGF-β1) expression and by diminishing the effects of pro-inflammatory cytokines.

High therapeutic concentration of prazosin up-regulates angiogenic IL6 and CCL2 genes in hepatocellular carcinoma cells.

Alteration of the oxidative stress of hepatocellular carcinoma (HCC) cells can influence the expressions of genes favored angiogenesis. Quinone reductase 2 which can activate quinones leading to reactive oxygen species production is a melatonin receptor known as MT3. Prazosin prescribed for benign prostate hyperplasia and hypertension is a potent antagonist for MT3. This study was to investigate the influence of therapeutic concentrations of prazosin (0.01 and 0.1μM) on cell proliferation and differential expressions of CCL2, CCL20, CXCL6, CXCL10, IL8 and IL6 genes related to inflammation and/or oxidative stress in human HCC cell lines. Two HCC cell lines including one without susceptible to amphotericin B-induced oxidative stress (cell line A; HCC24/KMUH) and one with this effect (cell line B; HCC38/KMUH) were investigated by 0.01 and 0.1μM prazosin. The premixed WST-1 cell proliferation reagent was applied for proliferation assay. Differential expressions of genes were examined by quantitative reverse transcriptase-polymerase chain reaction. Our results showed that both 0.01 and 0.1μM prazosin did not influence cell proliferation in both cell lines. Both 0.01 and 0.1μM prazosin in cell line A and 0.01μM prazosin in cell line B did not cause differential expressions of tested genes. However, 0.1μM prazosin caused remarkable up-regulation of IL6 gene and slightly up-regulation of CCL2 gene in cell line B. In conclusion, high therapeutic concentration of prazosin can up-regulate angiogenic IL6 and CCL2 genes in human HCC cells susceptible to amphotericin B-induced oxidative stress. Clinical application of prazosin in patients with HCC should consider this possibility.

Contrary influence of clinically applied sorafenib concentrations among hepatocellular carcinoma patients

The treatment responses of sorafenib in hepatocellular carcinoma are modest which may be due to different characteristics of cancer cells or insufficient therapeutic concentrations. This study was to clarify this issue. The anti-proliferative effects and differential expressions of 8 genes related to sorafenib anti-cancer mechanisms (tyrosine kinase receptor genes: KDR, PDGFRB; RAF cascade: RAF1, BRAF, MAP2K1, MAP2K2, MAPK1, MAPK3) were investigated in primary cultured hepatocellular carcinoma cells collected from 8 patients using clinically applied sorafenib concentrations (5, 10μg/mL). The anti-proliferative effects of sorafenib at either 5 or 10μg/mL, which were related to down-regulations of KDR, PDGFRB and/or genes in the RAF cascade, were achieved only in one patient (HCC38/KMUH). However, either 5 or 10μg/mL sorafenib promoted proliferation in 4 patients (HCC29/KMUH, HCC62/KMUH, HCC87/KMUH, HCC98/KMUH). Among them, the RAF cascade, PDGFRB and/or KDR were up-regulated in 3 patients but no gene was differentially expressed in the remaining one patient (HCC87/KMUH). Increase the sorafenib concentration to 10μg/mL paradoxically up-regulated and/or obliterated the previously down-regulated genes in the RAF cascade and/or KDR in 4 patients (HCC29/KMUH, HCC76/KMUH, HCC87/KMUH, HCC98/KMUH). Significant down-regulations of the RAF cascade and PDGFRB by sorafenib but without anti-proliferative effects were detected in one patient (HCC54/KMUH). In conclusion, influence of sorafenib on proliferation is not simply through the RAF cascade. The responses of KDR, PDGFRB and the RAF cascade to sorafenib among patients are diverse or even contrary. Increase the sorafenib concentration has potential to up-regulate genes favored angiogenesis and proliferation.

Expression of epidermal growth factor, basic fibroblast growth factor and insulin growth factor-1 and relation to myocyte regeneration of obstructed ureters in rats

Objective To investigate the roles of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and insulin growth factor-1 (IGF-1) in the myocyte regeneration of obstructed ureters. Material and methods The expression of EGF, bFGF, IGF-1 and proliferation cell nuclear antigen (PCNA) was studied immunohistochemically in 54 female Sprague–Dawley rats. Results Tissue damage to the smooth muscle layer in the obstructed ureters was aggravated during the period of obstruction. The expression of EGF, bFGF and IGF-1 in myocytes was detected using the method of concurrent immunohistochemical staining. The expression of EGF, bFGF and IGF-1 in the smooth muscle layer was found from Day 14 after ligation. The expression of EGF, bFGF and IGF-1 increased to a peak on Day 21 and then declined. The expression of PCNA in the smooth muscle layer was also found from Day 14 after ligation and increased to a peak on Day 21. The expressions of EGF, bFGF and IGF-1 were significantly correlated with the expression of PCNA in the smooth muscle layer (r=0.7982, 0.6264 and 0.5840, respectively; p-values all <0.002). Co-expression of EGF, bFGF, IGF-1 and PCNA was determined using the method of double immunofluorescence staining. Co-expression of PCNA was observed in 34% of EGF-positive myocytes, 53.6% of bFGF-positive myocytes and 41.1% of IGF-1-positive myocytes at Day 21 post-ligation. Conclusions Expression of EGF, bFGF and IGF-1 may contribute to myocyte regeneration of damaged ureters in rats with obstructive uropathy.