Relationship between CCL20/CCR6/Th17 axis and vascular invasion and metastasis in patients with primary hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the role of CCL20/CCR6/Th17 axis in vascular invasion and metastasis of primary hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Expression levels of CCL20 mRNA in the normal human liver cell line L-02, and human hepatocellular carcinoma cell lines Hep3B, Huh7 and HepG2 were quantified by using SYBR green real time PCR. CCL20 secretions from these cell lines were quantified by using ELISA. The chemotactic effect of HCC cell line Hep3B on human peripheral blood mononuclear cells was determined by using transwell chemotaxis assay. Pre-therapy serum levels of IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-17, IL-23, IFN-γ, TNF-α and CCL20 in 93 patients with HCC were measured by using 9-plex array and ELISA. All the patients were chronic hepatitis B virus associated HCC, and 51 cases were those with vascular invasion and metastasis (metastasis group) and 42 cases were not (non-metastasis group). CCL20 and CCR6 mRNA expressions in the HCC and tumor-adjacent tissues were determined by using SYBR Green real time PCR in 41 patients, among them, 20 cases were from the group of patients with metastasis and 21 cases were from the group of patients without metastasis. The CCL20 expression was further determined by immunohistochemistry.</p><p><b>RESULTS</b>The HCC cell lines expressed and secreted higher amount of CCL20, which effectively recruited CCR6(+) T cells. Pre-therapy serum levels of CCL20 in 93 HCC patients were (38.2 ± 28.4)pg/ml, significantly increased than those with benign hepatic hemangiomas [(7.8 ± 17.8)pg/ml, P < 0.01]. In addition, the serum levels of CCL20 were positively correlated with the tumor diameters in HCC patients (r = 0.32, P = 0.0018). CCL20 was dominantly expressed in the cytoplasm in HCC cells, and it was also expressed by some infiltrating immune cells. The mRNA expression levels of CCL20 of the tumor tissues were significantly higher than that in the tumor-adjacent tissues (P < 0.05). Multivariate logistic regression analysis showed that serum levels of IL-17 and CCL20 were independent risk factors of metastasis in HCC patients (P < 0.05 for both). CCL20 mRNA showed no statistically significant differences between patients with metastasis and without metastasis in both tumor tissues and tumor-adjacent tissues (P > 0.05 for both). But the patients with metastasis showed significantly higher expressions of CCR6 both in their tumor [5.75 (1.79, 19.13)]and tumor-adjacent tissues [7.99 (4.49, 19.54)] than those with non-metastasis [1.69 (0.76, 2.87) and 3.58 (1.84, 4.32), P < 0.05 for both].</p><p><b>CONCLUSION</b>CCL20/CCR6/Th17 axis may promote vascular invasion and metastasis hepatocellular carcinoma.</p>

Wnt/b-catenin signaling pathway affects the protein expressions of caspase-3, XIAP and Grp-78 in hepatocellular carcinoma / 中华肝脏病杂志

To investigate the relationship and significance of Wnt/b-catenin signaling pathway with caspase-3, XIAP, HSP27and Grp-78. The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against b-catenin. After 72 and 96 h, protein was extracted and the protein expressions of b-catenin, caspase-3, XIAP, Grp-78 and HSP27 were detected by Western blot. b-catenin protein expression was inhibited at both time points and the expression at 96 h was higher than that at 72 h (F = 160.72, P is less than to 0.01). Interestingly, Caspase-3 protein expression was decreased at 72 h and increased to normal at 96 h (F = 136.10, P is less than to 0.01), while p-caspase-3 protein expression increased at 72 h and decreased to normal at 96 h (F = 98.65, P is less than to 0.01). XIAP protein expression decreased at 72 h (F = 37.29, P is less than to 0.01) and increased at 96 h. Grp-78 protein expression increased at 72 h and decreased to normal at 96 h ( F = 58.72, P is less than to 0.01). HSP27 protein expression showed no change following transfection ( F = 1.91, P is more than to 0.05). Wnt/b-catenin signaling pathway is related to the protein expressions of caspase-3, XIAP and Grp-78, but not related to HSP27 protein expression in HCC. Wnt/b-catenin signaling pathway may participate in the regulation of HCC apoptosis, proliferation and differentiation through affecting these factors.

Different patterns of cyclin D1/CDK4-E2F-1/4 pathways in human embryo lung fibroblasts treated by benzoapyrene at different doses / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]P.</p><p><b>METHODS</b>Human embryo lung fibroblasts (HELFs) were treated with 2 micromol/L or 100 micromol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs). Cyclin D1, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle.</p><p><b>RESULTS</b>After B[a]P treatment, the proportion of the first gap (G1) phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 micromol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs (A-D1 and A-K4) and T-HELFs (T-A-D1 and T-A-K4). After 2 micromol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4.</p><p><b>CONCLUSIONS</b>Cyclin D1/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 micromol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 micromol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.</p>

Benzo (a) pyrene-induced human embryo lung cell cycle alterations through positive regulation of mitogen-activated protein kinase signal pathways / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.</p><p><b>METHODS</b>The phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.</p><p><b>RESULTS</b>The phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.</p><p><b>CONCLUSION</b>ERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.</p>

The activation of MAPK/cyclin D1-CDK4 signaling pathway caused by quartz / 中华劳动卫生职业病杂志

<p><b>OBJECTIVES</b>To study the phosphorylation level of mitogen activated protein kinase (MAPK) in human embryonic lung fibroblasts (HELF), and the expression level of cyclin D1-CDK4 protein in S-HELF and whether the expression level of cyclin D1-CDK4 protein mediated by MAPK/AP-1 signaling pathway in S-HELF.</p><p><b>METHODS</b>Two kinds of treatment: (1) Cells were harvested after stimulation 2 h for the detection of cytokines. (2) Cells were stimulated by quartz for a long time (2 months) for transformation characters (S-HELF). The MAP kinase was detected by western blot. Cyclin D1 and CDK4 (cyclin dependent kinase 4) proteins was measured by immunocytochemistry. Flow cytometry was used to evaluate the alternation of cell cycle.</p><p><b>RESULTS</b>Crystalline quartz could cause the phosphorylation level of ERKs, p38K, and JNKs in HELF increase. However, activated levels of ERKs and p46 of JNKs increased in S-HELF, and p38K activation decreased, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Cyclin D1 and CDK4 protein expression levels increased in S-HELF as compared with parental HELF. Inhibition of ERKs activation by AG126, AP-1 by curcumin, and JNKs by SP600125 could reduced the induction of cyclin D1 and CDK4, whereas inhibition of p38K by SB203580 did not show any inhibitory effects on S-HELF.</p><p><b>CONCLUSIONS</b>The phosphorylation levels of ERK1/2, JNK1/2, and p38 increased in HELF exposed to quartz. The phosphorylation levels of ERK1/2 and JNK1 increased, but the phosphorylation level of p38 decreased in S-HELF. The expression level of cyclin D1-CDK4 protein increased in S-HELF. Overexpression of cyclin D1-CDK4 is due to the activation of ERKs, JNKs/AP-1 signaling pathway in S-HELF.</p>

Vitamin C reverses benzo (a) pyrene-induced cell cycle changes by E2F pathway / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the role of E2F1/4 pathway in vitamin C reversing benzo (a) pyrene [B (a) P]-induced changes of cell cycle in human embryo lung fibroblasts (HELF) and the relationship between E2F1 and cyclin D1/CDK4.</p><p><b>METHODS</b>The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established to detect the relationship of signaling pathway. Cells were cultured and pretreated with vitamin C before stimulation with B (a) P for 24 hours. The expression levels of cyclin D1, CDK4, E2F1 and E2F4 were determined by Western blot and the band intensity was analysed as the relative value to control by using the Gel-Pro 3.0 software. Flow Cytometric Analysis was employed to detect the distributions of cell cycle.</p><p><b>RESULTS</b>B (a) P significantly elevated the expression levels of cyclin D1, CDK4, E2F1 and E2F4 in HELF cells. Vitamin C decreased the expression levels of above proteins in B (a) P-stimulated HELF cells. The expression levels of these proteins in B (a) P-treated above transfectants were lower than those in B (a) P-treated HELF cells. The expression levels of above proteins with vitamin C combined with antisense cyclin D1 were decreased as compared to those with antisense cyclin D1 alone. B (a) P increased the percentage of S phase as compared to the controls [(41.1 +/- 0.2)% vs (33.5 +/- 3.2)%, P < 0.05]. Both vitamin C [(33.2 +/- 0.6)% vs (41.1 +/- 0.2)%, P < 0.05] and antisense cyclin D1 [(31.2 +/- 1.3)% vs (41.1 +/- 0.2)%, P < 0.05] suppressed the changes of cell cycle induced by B (a) P. Vitamin C combined with antisense CDK4 markedly suppressed B (a) P-induced changes of cell cycle as compared to those with antisense CDK4 alone.</p><p><b>CONCLUSION</b>Vitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.</p>

Vitamin C inhibits benzoapyrene-induced cell cycle changes partly via cyclin D1/E2F pathway in human embryo lung fibroblasts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELF) cells.</p><p><b>METHODS</b>The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle.</p><p><b>RESULTS</b>B[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C (10, 100, 500, 1000, and 5000 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone.</p><p><b>CONCLUSIONS</b>B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.</p>

cyclin D1/E2F pathways involved in cell cycle changes of human embryo lung fibroblasts induced by benzo(a)pyrene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the roles of cyclin D1/CDK4-E2F-1/4 pathway in cell cycle changes of human embryo lung fibroblasts (HELF) induced by two different benzo(a)pyrene [B(a)P] treatment models.</p><p><b>METHODS</b>Two B(a)P treatment models: HELF were treated by 2 micromol/L B(a)P for 24 hours; HELF were treated by 100 micromol/L B(a)P three times 24 hours each and provide with some characteristics of transformed cells (T-HELF). Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometry and Western bolt analysis.</p><p><b>RESULTS</b>After 24 hours 2 microml/L B(a)P treatment, the HELFs in the G(1) phase was decreased. In HELF transfected with antisense cyclin Dl (A-Dl) and antisense CDK4 (A-K4), the expression of cyclin Dl and CDK4 blocked the cell cycle changes from the G(1) phase to the S phase induced by B(a)P. The overexpression of cyclin Dl and E2F-1 in HELF was induced by B(a)P. The E2F-1 overexpression in A-D1 induced B(a)P was inhibited. The E2F-4 expression was decreased and the CDK4 expression was increased significantly in A-K4 after B(a)P treatment. Most of T-HELF transfected with antisense cyclin Dl (T-A-Dl) and antisense CDK4 (T-A-K4) were retained in G(1) phase. The cyclin Dl expression in T-HELF was increased significantly compared with that in HELF. The E2F-4 expression in T-A-Dl and T-A-K4 was increased significantly compared with that in T-HELF.</p><p><b>CONCLUSION</b>B(a)P induces the cell cycle changes through cyclin D1/CDK4-E2F-1/4 pathway in HELF treated by 2 micromol/L B(a)P while it induces cell cycle changes through cyclin D1/CDK4-E2F-4 pathway in T-HELF.</p>

Inhibition of the pathway of benzo (a) pyrene-induced cell cycle changes by all-trans retinoic acid in lung fibroblast / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the reverse effect of all-trans retinoic acid (ATRA) on Benzo (a) pyrene (B (a) P)-induced cyclin D1, CDK4, E2F-1 and E2F-4 expression and cell cycle progression in human embryo lung fibroblast (HELF).</p><p><b>METHODS</b>After HELF cells was treated with ATRA, they were exposed to 2 micromol/L of B (a) P. Western blotting was employed to detect protein expression level; the RNA transfection techniques was used to investigate ATRA-induced signal pathway; flow cytometry was used to detect cell cycle progression.</p><p><b>RESULT</b>After treatment with 2 micromol/L B (a) P for 24 h, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF; the expression of E2F-4 and CDK4 were not changed markedly; pretreatment with 0.1 micromol/L ATRA for 24 h could efficiently decrease B (a) P-induced overexpression of cyclin D1 and E2F-1; stimulation to antisense cyclin D1 or antisense CDK4 by B (a) P could significantly impair E2F-1 up-regulation; pretreatment with ATRA, cells with antisense cyclin D1 or antisense CDK4 showed a less decrease in B (a) P-induced overexpression of E2F-1 compared to similarly treated control cells; flow cytometry analysis showed B (a) P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B (a) P-induced cell cycle progression by an accumulation of cells in the G(1) phase.</p><p><b>CONCLUSION</b>ATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.</p>