CAF-derived midkine promotes EMT and cisplatin resistance by upregulating lncRNA ST7-AS1 in gastric cancer.

This study aimed to investigate the role of cancer-associated fibroblast (CAF)-derived midkine (MK) in cisplatin (DDP) resistance. The primary cultures of CAFs and non-cancer fibroblasts (NFs) were isolated and purified. The DDP-resistant gastric cancer (GC) cells were cultured with CAF-conditioned medium. QRT-PCR and Elisa assays were employed to determine MK expression. The expression of ST7-AS1 was measured by qRT-PCR. The impact of CAFs, MK, and ST7-AS1 silencing on DDP resistance was determined by MTT and Annexin V/PI staining assay. Expression of EMT markers and PI3K/AKT was determined by Western blot and qRT-PCR. The role of MK in DDP resistance was confirmed in a xenograft model. Incubation with CAF-conditioned medium increased the IC50 to DDP. Also, incubation with CAF-conditioned medium increased cell viability, reduced cell apoptosis, and promoted EMT in DDP-resistant GC cells, which were all blocked with MK neutralization antibody treatment. MK increased the DDP resistance and upregulated the expression of ST7-AS1 in DDP-resistant GC cells. Additionally, ST7-AS1 knockdown increased the sensitivity to DDP by inhibiting EMT. Moreover, ST7-AS1 knockdown significantly decreased the phosphorylation of PI3K and AKT, and suppressed EMT, which were restored by MK addition. Finally, MK promoted tumor growth and DDP resistance in a mice model bearing the SGC-7901/DDP xenografts. CAF-derived MK promotes EMT-mediated DDP resistance via upregulation of ST7-AS1 and activation of PI3K/AKT pathway.

CircN4BP2L2 promotes colorectal cancer growth and metastasis through regulation of the miR-340-5p/CXCR4 axis.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. Dysregulation of circular RNAs (circRNAs) appears to be a critical factor in CRC progression. However, mechanistic studies delineating the role of circRNAs in CRC remain limited. In this study, qRT-PCR and western blot assays were used to measure the expression of genes and proteins. Migration, invasion, proliferation, and apoptosis were examined by wound-healing, transwell, CCK-8, colony formation, and flow cytometry assays, respectively. Molecular interactions were validated by a dual-luciferase report system. A xenograft animal model was established to examine in vivo tumor growth and lung metastasis. Our data indicated that circN4BP2L2 expression was increased in CRC tissues and cell lines. Notably, inhibition of circN4BP2L2 effectively inhibited proliferation, migration, and invasion of LoVo cells, and inhibited tumor growth and metastasis in vivo, whereas the forced expression of circN4BP2L2 facilitated the proliferation, migration, and invasion of HT-29 cells. Mechanistic studies revealed that circN4BP2L2 acted as a molecular sponge of miR-340-5p to competitively promote CXCR4 expression. Furthermore, inhibition of miR-340-5p reversed the anti-cancer effects of circN4BP2L2 or CXCR4 silencing. Our data indicated an oncogenic role of circN4BP2L2 in CRC via regulation of the miR-340-5p/CXCR4 axis, which may be a promising biomarker and target for CRC treatment.

Overexpression of ANLN contributed to poor prognosis of anthracycline-based chemotherapy in breast cancer patients.

PURPOSE: To investigate the associations of ANLN expression with prognosis of breast cancer and clinical outcome of anthracycline-based chemotherapy. METHODS: This study enrolled 308 breast cancer patients in which 264 of them received anthracycline-based chemotherapy. Immunohistochemistry was used to detect ANLN expression level of the patients. Clinical characteristics of the patients were collected, and associations of ANLN expression with prognosis were analyzed. RESULTS: Our results showed that ANLN expression was associated with survival of breast cancer patients, and it was also related to clinical outcome of patients received anthracycline-based chemotherapy. Breast cancer patients with high expression of ANLN would have poor prognosis and poor clinical outcome to anthracycline-based chemotherapy. CONCLUSION: ANLN could be an independent prognosis predictor for breast cancer, and its expression might be used to predict the anthracycline-based chemotherapy clinical outcome in breast cancer patients.

GPC-1 may serve as a predictor of perineural invasion and a prognosticator of survival in pancreatic cancer.

OBJECTIVE: The purpose of this study was to investigate the expression of glial cell derived neurotrophic factor (GDNF), glypican-1 (GPC-1), and matrix metalloproteinase-9 (MMP-9), and their association with clinicopathologic characteristics as well as prognostic significance in pancreatic cancer. METHODS: Immunohistochemical assessment of GDNF, GPC-1, and MMP-9 was performed in 62 cases of surgically resected pancreatic cancer. Perineural invasion in pancreatic cancer was observed by marking nerve fiber with S-100, while 16 normal pancreatic tissues were used as normal control. Correlations of GDNF, GPC-1 and MMP-9 expressions with clinicopathologic parameters were analyzed. A survival analysis was performed to find the prognostic significance. RESULTS: The expressions of GDNF, GPC-1 and MMP-9 in pancreatic cancer tissue were significantly higher than of those in normal pancreatic tissues (41/62 vs. 5/16 for GDNF, 35/62 vs. 2/16 for GPC-1, and 37/62 vs. 3/16 for MMP-9; p<0.01, respectively). The overexpression of GDNF, GPC-1, and MMP-9 in pancreatic cancer tissue was significantly related to the perineural invasion (p<0.05). Although the overexpression of these genes was related to poor survival, GPC-1 had an independent prognostic effect on overall survival. CONCLUSION: GPC-1 is significantly related to the perineural invasion of pancreatic cancer, holding some prognostic significance in patients with pancreatic cancer.

Detection of STAT2 in early stage of cervical premalignancy and in cervical cancer.

OBJECTIVE: To measure the expression pattern of STAT2 in cervical cancer initiation and progression in tissue sections from patients with cervicitis, dysplasia, and cervical cancer. METHODS: Antibody against human STAT2 was confirmed by plasmids transient transfection and Western blot. Immunohistochemistry was used to detect STAT2 expression in the cervical biopsies by using the confirmed antibody against STAT2 as the primary antibody. RESULTS: It was found that the overall rate of positive STAT2 expression in the cervicitis, dysplasia and cervical cancer groups were 38.5%, 69.4% and 76.9%, respectively. The STAT2 levels are significantly increased in premalignant dysplasia and cervical cancer, as compared to cervicitis (P< 0.05). Noticeably, STAT2 signals were mainly found in the cytoplasm, implying that STAT2 was not biologically active. CONCLUSIONS: These findings reveal an association between cervical cancer progression and augmented STAT2 expression. In conclusion, STAT2 increase appears to be an early detectable cellular event in cervical cancer development.

Effect of eIF3a on response of lung cancer patients to platinum-based chemotherapy by regulating DNA repair.

PURPOSE: The purpose of this study is to test the hypothesis that eIF3a may regulate the expression of DNA repair proteins which, in turn, affects response of lung cancer patients to treatments by DNA-damaging anticancer drugs. EXPERIMENTAL DESIGN: Immunohistochemistry was used to determine the expression of eIF3a in 211 human lung cancer tissues followed by association analysis of eIF3a expression with patient's response to platinum-based chemotherapy. Ectopic overexpression and RNA interference knockdown of eIF3a were carried out in NIH3T3 and H1299 cell lines, respectively, to determine the effect of altered eIF3a expression on cellular response to cisplatin, doxorubicine, etoposide (VP-16), vincristine, and vinblastine by using MTT assay. The DNA repair capacity of these cells was evaluated by using host-cell reactivation assay. Real-time reverse transcriptase PCR and Western Blot analyses were carried out to determine the effect of eIF3a on the DNA repair genes by using cells with altered eIF3a expression. RESULTS: eIF3a expression associates with response of lung cancer patients to platinum-based chemotherapy. eIF3a knockdown or overexpression, respectively, increased and decreased the cellular resistance to cisplatin and anthrocycline anticancer drugs, DNA repair activity, and expression of DNA repair proteins. CONCLUSIONS: eIF3a plays an important role in regulating the expression of DNA repair proteins which, in turn, contributes to cellular response to DNA-damaging anticancer drugs and patients' response to platinum-based chemotherapy.

The effects of hepatitis C virus non-structural protein 3 on cell growth mediated by extracellular signal-related kinase cascades in human hepatocytes in vitro.

Hepatitis C virus (HCV) infection has become a severe health problem worldwide. The viral proteins are believed to be among the most important factors that contribute to HCV mediated pathogenesis. Accumulated evidence demonstrating that HCV non-structural protein 3 (NS3) possesses oncogenic potential, and is involved in the regulation of cell proliferation has been documented. In this study, we emphasized the effect of HCV NS3 protein on cell proliferation in the immortally normal hepatocyte QSG7701 cells. The cell line transfected with plasmid expressing NS3 protein showed enhanced cell growth, extracellular signal-related kinase (ERK) activation, DNA binding activities of transcription factors of activator protein 1 (AP-1) and NF-kappaB, and cyclin D1 overexpression, but without activation of Jun amino-terminal kinase or p38. Pre-treatment of NS3 protein expressing cells with ERK inhibitor, PD98059, blocked the activation of AP-1 and NF-kappaB, and inhibited cyclin D1 expression and cell proliferation. The results suggest that NS3-mediated cell growth occurs through activation of ERK/AP-1 and NF-kappaB/cyclin D1 cascades.

Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein: a potential for developing hepatitis C virus targeting gene therapy.

AIM: To examine whether 2'-5'oligoadenylate synthetase (OAS) gene promoter can be specifically activated by hepatitis C virus (HCV)-core protein. METHODS: Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. RESULTS: L02/core cell line stably expressing HCV-core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION: HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

HSP70 and mucin 5B: novel protein targets of N,N'-dinitrosopiperazine-induced nasopharyngeal tumorigenesis.

N,N'-Dinitrosopiperazine (DNP) induces nasopharyngeal carcinoma (NPC) and shows organ specificity to the nasopharyngeal epithelium. To investigate its mechanism, the rat NPC model was induced using DNP. Rat NPC and normal nasopharyngeal cells were obtained from the NPC model using laser capture. The total proteins from these cell samples were separated with two-dimension polyacrylamide gel electrophoresis techniques, and highly expressed proteins (> five-fold) were analyzed using matrix-assisted laser desorption/ionization time of flight and bioinformatics. The results showed that HSP70 and mucin 5B expression increased not only in rat NPC but also in atypical hyperplasia nasopharyngeal tissues, a precancer stage of NPC. High-expression of heat shock protein 70 (HSP70) and mucin 5B was further supported by western blot analysis. The immunofluorescence and western-blotting studies further showed that DNP induced the expression of HSP70 and mucin 5B in a dosage-dependent manner in normal nasopharyngeal epithelia cells. Our data indicate that DNP triggers over-expression of HSP70 and mucin 5B, and is involved in nasopharyngeal tumorigenesis. HSP70 and mucin 5B may be important targets in nasopharyngeal tumorigenesis induced by DNP.

[AP-1 regulates TGF-beta1-induced secretion of Type I collagen in human lung fibroblasts].

OBJECTIVE: To investigate the role of AP-1 in the secretion of Type I collagen in TGF-beta1-stimulated human lung fibroblasts. METHODS: Human lung fibroblasts cell line (HLF-02) was cultured, and then stimulated with 10 microg/L TGF-beta1 at different time points. Curcumin was added into the culture medium to inhibit the AP-1 activity before incubating with TGF-beta1. AP-1 DNA binding activity was assayed by electrophoretic mobility shift assay (EMSA), and the expression of Type I collagen was detected by Western blot and RT-PCR. RESULTS: TGF-beta1 could induce the transcription and secretion of Type I collagen in HLF-02 cells(P<0.05). TGF-beta1 could upregulate the AP-1 DNA binding activity ( P<0.05). Curcumin ( 5, 10, 15, and 20 micromol/L) could inhibit the AP-1 DNA binding activity in TGF-beta1-stimulated cells (the inhibition ratio was 17.1%, 17.6%, 24.2%, and 31.3%; P<0.05). Curcumin (5, 10, 15, and 20 micromol/L) could also inhibit the secretion of Type I collagen significantly (the inhibition ratio was 62.1%, 58.8%, 62.1%, and 59.6%; P<0.05). CONCLUSION: AP-1 is responsible for the secretion of TGF-beta1-induced Type I collagen in human lung fibroblasts.

[Mechanism of hepatocyte transformation by HCV NS3 using two-dimensional electrophoresis and mass spectrometry].

OBJECTIVE: To investigate the proteome of hepatocyte transformation by hepatitis C virus (HCV) nonstructural protein 3 (NS3). METHODS: Human hepatocyte line QSG7701 stably expressing HCV NS3 C-terminal deleted protein was constructed, which was named pRcHCNS3/QSG. Two-dimensional electrophoresis (2-DE) was used to separate the total protein of pRcHCNS3/QSG and pRcCMV transfected cells (pRcCMV/QSG) respectively. Differentially expressed protein spots were identified by mass spectrometry. Western blot confirmed the differentially expressed proteins. RESULTS: 2-DE profiles with high resolution and reproducibility were obtained. The average spots of pRcHCNS3/QSG and pRcCMV/QSG were (1183+/-77) and (1095+/-82) respectively, and (920+/-60) spots were matched. Twenty-one differentially expressed protein spots were chosen randomly and 15 were identified by mass spectrometry. Some proteins such as Ras, P38 and HD53 which were involved in signal transduction were increased in pRcHCNS3/QSG cells. Western blot also showed strong expression of phosphorylated P44/42 and P38 in pRcHCNS3/QSG cells. Other differentially expressed proteins were related to cell cycle regulation, immunoreaction, tumor invasion and metastasis, and liver metabolizability. CONCLUSION: HCV NS3 might be involved in cell malignant transformation through affecting protein expression and signal transduction such as MAPK cascade. Further study on the signal transductions and their relationship would not only be helpful to explore the mechanism of HCV related HCC, but also provide a new idea for the molecular treatment of HCC.

[Cross-talk between ERK and NF-kappaB signal transduction pathways in the hepatocytes expressing hepatitis C virus nonstructural protein 3].

OBJECTIVE: To explore the cross-talk between extracellular signal-regulated kinase (ERK) and nuclear factor (NF-kappaB) signal transduction pathways in the hepatocytes expressing hepatitis C virus nonstructural protein 3 (HCV NS3). METHODS: A cell line QSG7701/NS3, which stably expressed HCV NS3 protein, was constructed by transfecting plasmid pcDNA3.1-NS3 into a human immortalized hepatocyte line QSG7701. Before and after QSG7701/NS3 cells were treated by MEK inhibitor PD98059, the phosphorylation level of ERK and the expression of cyclin D1 protein were detected by Western blot; the DNA binding activities of activator protein 1 (AP-1) and NF-kappaB were evaluated with electrophoretic mobility shift assay (EMSA). Cell cycles were measured by flow cytometry (FCM); the effects of PD98059 on the cell proliferation were determined by MTT assay. RESULTS: HCV NS3 protein could up-regulate the phosphorylation of ERK and the expression level of cyclin D1 in QSG7701 cells. PD98059 could inhibit the cell proliferation mediated by HCV NS3 protein, down-regulate the activities of AP-1 and NF-kappaB, and suppress the expression of cyclin D1. CONCLUSION: The inhibition of ERK can suppress the DNA binding activity of NF-kappaB and the cell proliferation mediated by HCV NS3 protein in a dose-dependent manner. There may be a cross-talk between ERK and NF-kappaB signal transduction pathways, which may exert synergistic action on the proliferation and malignant transformation of hepatocytes.

[Expression and role of nuclear transcription factor Sp1 in macrophages stimulated by silicon dioxide].

OBJECTIVE: To study the expression and localization of nuclear transcription factor Sp1 in macrophages after stimulated by silicon dioxide in vivo and in vitro. METHODS: Forty Sprague Dawley rats were randomly divided into the control group and the silica exposure group, 20 in each group. The rat silicosis models were established by direct tracheal instillation of silica into rat lung (0.2 g/kg) only once while the control group was instilled with equal amount of saline. Animals were killed at 1st, 7th, 14th, 21st and 28th day after instillation. Dynamic changes of Sp1 protein expression and its cellular localization were detected by immunohistochemistry in pulmonary macrophages. In vitro, Sp1 mRNA and protein expression and their dynamic changes were monitored by RT-PCR and western blotting after stimulated by silicon dioxide in cultured RAW264.7 macrophages respectively. Cellular localization of Sp1 protein was characterized by immunocytochemistry. RESULTS: Compared to the control group, the Sp1 protein expression was increased in pulmonary macrophages and reached the peak at the 14th day in the silica exposure group. In vitro, the Sp1 mRNA level began to rise at 30 minutes after the administration of silicon dioxide and reached the peak at 240 minutes and then decreased to the minimal level at 960 minutes. The Sp1 total protein and nuclear protein also exhibited the similar trend. The former reached the peak at 240 minutes and the latter at 480 minutes. The significant nuclear translocation of Sp1 protein was observed at 120 minutes after the administration of silicon dioxide and became most significant at 480 minutes. CONCLUSION: Silicon dioxide can activate nuclear transcription factor Sp1 in macrophages in vivo and in vitro. Sp1 might play an important pathogenic role in the development of silicosis.

[Expression of Ang2 and Tie2 and their relation with the angiogenesis of hepatocellular carcinoma in rats].

OBJECTIVE: To investigate the relationship between the expression of Ang2, Tie2 and the angiogenesis of hepatocellular carcinoma in rats. METHODS: Thirty-eight healthy male rats were randomly divided into 3 groups: 5 rats in the control group; 25 rats in the experimental group were equally divided into 5-day, 10-day, 15-day, 20-day, and 25-day groups; the other 8 rats were used as the supplement of the experimental group. An allogenic transplanted rat model of CBRH-7919 hepatocellular carcinoma in situ was established by immunosuppression. The expressions of Ang2 and Tie2 were detected by immunohistochemical staining in cancerous tissues of different developmental stages and liver tissues of the control group. At the same time, microvessel density was determined by anti-CD31 immunohistochemical staining. RESULTS: CBRH-7919 hepatocellular carcinoma models were successfully set up in 24 rats. The expression level of Ang2 and Tie2 in cancerous tissues was much higher than that of liver tissues of the control group (P <0.05). The overexpression of Ang2 was pristine and continuous in different developmental stages. The expressions of Ang2 and Tie2 positively correlated with microvessal density in hepatocellular carcinoma (P<0.05). CONCLUSION: The up-regulation of Ang2 and Tie2 may play important roles in the angiogenesis of hepatocellular carcinoma. Ang2 may participate in the start of angiogenesis of hepatocellular carcinoma.

[p38/ERK signal pathways regulating the expression of type I collagen and activity of MMP-2 in TGF-beta1-stimulated HLF-02 cells].

OBJECTIVE: To investigate the role of TGF-beta(1)/MAPK signaling pathways in the expression of type I collagen and activity of MMP-2, 9 in human lung fibroblasts. METHODS: Human lung fibroblasts cell line (HLF-02) was cultured and and then stimulated with 10 ng/ml TGF-beta(1) for different time; SB203580 or PD98059 was added into culture medium to block p38 or ERK kinase pathway before incubated with TGF-beta(1); the expression of type I collagen was detected by Western blotting and RT-PCR; zymogram analysis was used to analyze the activity of MMP-2 and MMP-9. RESULTS: (1) In the process of stimulation by TGF-beta(1), the type I collagen mRNA level of 24 h, 48 h and 72 h group was: 1.33 +/- 0.07, 2.46 +/- 0.09 and 2.39 +/- 0.08 respectively; and the type I collagen protein level of 24 h, 48 h and 72 h group was: 114.89 +/- 8.95, 208.16 +/- 6.75 and 211.46 +/- 8.05 respectively; and the activity of MMP-2 of 24 h, 48 h and 72 h group was: 190.33 +/- 5.86, 214.33 +/- 8.39 and 212.67 +/- 11.59 respectively. (2) SB203580 significantly inhibited the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 51%, 24% and 20%); (3) PD98059 also significantly attenuated the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 42%, 13% and 16%). CONCLUSION: TGF-beta(1) is capable of inducing the expression of type I collagen mRNA and protein and up-regulating MMP-2 activity in HLF-02 cells. p38 and ERK kinase signaling pathways play important role in regulation and control for this process.

[Effect of erigeron breviscapus on the expression of vascular endothelial growth factor in the periodontal tissues of rabbits during orthodontic tooth movement].

OBJECTIVE: To detect the effect of erigeron breviscapus on the expression of vascular endothelial growth factor (VEGF) in the periodontal tissues during orthodontic tooth movement. METHODS: 45 rabbits were divided into 3 groups (groups A, B and C). Groups A and B included experimental group of 1, 3, 7 and 14 days respectively. The mandibular first molar of each experimental rabbit was observed. The rabbits of group A and group B received iontophoresis with erigeron breviscapus into the right (group A-R and group B-R) and with normal sodium into the left as the control (group A-L and group B-L). Additionally, the rabbits of group B were designed orthodontic appliance, by which 0.78 N mesial force was applied to pull the mandibular first molars. Group C, group of 0 day, was no iontophoresis and orthodontic appliance as the control. After killed on schedule, the amount of experimental tooth movement was measured and the expression of VEGF was examined by immunohistochemical method. RESULTS: The amount of experimental tooth movement increased successively from 1 to 14 days. The differences among days 3, 7 and 14 were significant in the comparison between group B-R and group B -L (P < 0.01). The expression of VEGF in groups A-R and B-L enhanced apparently compared with that in groups C and A-L (P < 0.01), but that in group B-R was the most apparent (P < 0.01). The expression of VEGF reached the peak level on day 3 in groups A-R and B-R (P < 0.01), but it reached the peak level on day 7 in group B-L (P < 0.01). CONCLUSION: Erigeron breviscapus by iontophoresis can accelerate orthodontic tooth movement, and can meanwhile up-regulate the expression of VEGF in periodontium in the earlier period of orthodontic tooth movement. Thus it can be presumed that one of its mechanisms for erigeron breviscapus to accelerate orthodontic tooth movement is erigeron breviscapus effects the metabolism and differentiation of osteoblast and osteoclast through up-regulating the expression of VEGF in periodontium.

[Effects of silica on the expression of plasminogen activator inhibitor-1 and activator protein-1 in human alveolar epithelial cells type II].

OBJECTIVE: To investigate the mechanism of silicosis by observing the effects of silica on the expression of plasminogen activator inhibitor-1 (PAI-1) and activator protein-1 (AP-1) in human alveolar epithelial cells type II (A549). METHODS: A549 cell and SiO(2) (200 microg /ml) were co-cultured for 0, 4, 8, 16 and 24 h respectively. The reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and SP immunocytochemistry were used for detections of the PAI-1 mRNA and protein expression. The nucleoprotein and total protein expression of AP-1 were investigated by Western blotting. RESULTS: The expression levels of PAI-1 mRNA and protein were increased in a time-dependent manner(r(mRNA) = 0.911, r(protein) = 0.902, P < 0.05). The expressions of PAI-1 mRNA and protein in experimental groups were higher than that in control group (P < 0.05) and was the highest in 24 h group [(0.73 +/- 0.01) vs (0.36 +/- .03)]. The nucleoprotein expressions of c-jun/c-fos in experimental groups were also higher than in control group (P < 0.05), and the nucleoprotein expression level of c-jun was the highest in 4 h group [(1.54 +/- 0.02) vs (0.56 +/- 0.03)]; the nucleoprotein expression level of c-fos was the highest in 8 h group [(0.36 +/- 0.01) vs (0.15 +/- 0.01)]. Both c-jun and c-fos expression were decreased after 16 h, but the total protein expression of c-jun/c-fos had no difference in all experimental groups. The positive signal of PAI-1 was located in cytoplasm and nucleus. CONCLUSION: SiO(2) could induce PAI-1 expression of A549 in a time-dependent manner, and AP-1 activation can be observed in early time.

[Apoptosis and caspase-3 in the model of rat silicosis].

OBJECTIVE: To explore the changes of apoptosis of cells in the lung tissue of rats with silica instillation and to its significance in silicosis, and to clarify the role of caspase-3 in the apoptosis progress. METHODS: Forty-eight rats were randomly divided into saline control groups and silica instillation groups, and the silicosis model was established in rats. Flow cytometry was used for detecting the rate of apoptosis at various stages. Immunohistochemistry for the expression of cleaved caspase-3. RESULTS: The model of rat silicosis was established successfully. The apoptosis rate in the experimental group was significantly higher than that in the control group, and was increased with time. Caspase-3 was mainly expressed in alveolar epithelium cells, pulmonary macrophages and infiltrated inflammation cells. The expression of caspase-3 in the experimental group was stronger than that in the control group, but its expression intensity was not related to the cell apoptosis (r = 0.215, P > 0.05). CONCLUSION: The apoptosis of the lung cells plays an important role during rat silicosis genesis. Caspase-3 plays an important role in regulating cell apoptosis during rat silicosis genesis.

[Transforming growth factor-beta1 induced phenotypic differentiation of human lung fibroblasts through mitogen activated protein kinase-dependent pathway].

OBJECTIVE: To investigate the role of MAPK signal transduction in TGF-beta1 induced phenotypic differentiation of human lung fibroblasts. METHOD: Human lung fibroblasts cell line (HLF-02) were cultured and then stimulated with 10 ng/ml TGF-beta1 for different time; SB203580 or PD98059 was added into culture medium to prevent p38 or Erk kinase pathway before incubating with TGF-beta1; the expression of alpha-smooth muscle actin (alpha-SMA) was detected by Western blotting and RT-PCR; Western blotting was used to assay phosphorylation of p38, Erk, and JNK kinase. RESULTS: (1) In the process of stimulation by TGF-beta1, the alpha-SMA mRNA expression levels of 24, 48 and 72 h groups were 1.87 +/- 0.11, 2.49 +/- 0.10, 3.02 +/- 0.15 respectively; and the alpha-SMA protein expression levels of 24, 48 and 72 h groups were 3.20 +/- 0.14, 3.96 +/- 0.21, 4.57 +/- 0.13 respectively. (2) TGF-beta1 induced p38, Erk kinase phosphorylation but not JNK kinase. (3) The inhibitors SB203580 and PD98059 suppressed TGF-beta1-induced p38 kinase and Erk phosphorylation respectively. (4) SB203580 significantly attenuated TGF-beta1-induced alpha-SMA mRNA and protein expression (inhibition rate: 30% and 40%); PD98059 also significantly inhibited TGF-beta1-induced alpha-SMA mRNA and protein expression (inhibition rate: 10% and 20%). CONCLUSION: TGF-beta1 is capable of inducing the phenotypic differentiation of HLF-02, which is regulated by p38 and Erk kinase signal pathway.