Objective To explore the expression of transporter associated with antigen processing (TAP) and major histocompatibility complex class (MHC)-Ⅰ molecule in malignant melanoma cell lines. Methods Three malignant melanoma cell lines, including A375, A875, and KZ28 cells as well as normal melanocytes were cultured. Western blot, reverse transcription PCR and flow cytometry were used to detect the protein and mRNA expression of TAP as well as the membrane expression of MHC-Ⅰ in these cells. Results A significant decrease was observed in the expression of TAP mRNA (t = 5.89, 4.45 and 4.57 re-spectively, all P< 0.01) and protein (t= 5.46, 4.32 and 4.67 respectively, all P< 0.01) in A375, A875 and KZ28 cells compared with the melanocytes, with the strongest decrease occurring in A375 cells. Similarly, the expression of MHC-Ⅰ molecule was significantly lower in A375, A875 and KZ28 cells than that in the melanocytes (t= 6.16, 5.22 and 5.61 respectively, all P< 0.01).Conclusions The protein and mRNA expres-sion of TAP is down-regulated in three melanoma cell lines A375, A875 and AZ28, which may contribute to the escape of melanoma cells from human immune surveillance.

Objective To investigate the expression and distribution of cellular FLICE-inhibitory protein (c-FLIP) in peripheral blood and lesions of psoriatic patients. Methods Peripheral blood and skin samples were obtained from 30 patients with psoriasis vulgaris and 20 normal controls. Flow cytometry was used to detect intracellular c-FLIP protein in peripheral T and B lymphocytes, immunohistochemistry to examine the expression of c-FLIP in lesional tissue. Results Based on the positivity rate of c-FLIP, there was a significant increase in T lymphocytes in active psoriasis compared with regressive psoriasis and normal controls (6.32%±1.17% vs 2.64%±0.74% and 2.28%±0.54%, P＜0.01 and 0.05, respectively), while no significant difference was found in B lymphocytes among these three groups (0.78%±0.16%, 0.71%±0.32%, 0.69%±0.18%, respectively, P＞0.05). The expression intensity of c-FLIP in keratinocytes was also higher in active psoriasis than in regressive psoriasis and normal controls (89.73±5.24 vs 117.40±7.50,121.58±7.93, P＜0.01 and 0.05 respectively), and there was no difference between regressive psoriasis and normal controls (P＞0.05). Conclusions c-FLIP is highly expressed in lesions and peripheral T lymphocytes of patients with active psoriasis, suggesting the possible involvement of c-FLIP in the proliferation of T lymphocytes in psoriasis.

Objective To study the expression of cellular FLICE inhibitory proteins(c-FLIP)in peripheral blood B lymphocytes in patients with systemic lupus erythematosus (SLE)and its correlation with clinical features.Methods Blood samples were obtained from 53 patients with SLE and 30 normal human controls.Flow cytometry and ELISA were performed to measure the expression of c-FLIP in pefipheral blood B lymphocytes and serum levels of IL-4 and IL-10,respectively.Relevant laboratory examinations were carried out for these patients.SLE disease activity index(SLEDAI)score was calculated for patients.Results The positivity rate of c-FLIP in B lymphocytes was 3.11%±0.70%in 18 patients with active SLE.significantly higher than that in 35 patients with inactive SLE (0.78%±0.28%)and normaI controls(0.68%±0.12%),while no statistical difierence was found between inactive patients and controls(t=1.56,P＞0.05).In SLE patients,the expression of c-FLIP showed a positive correlation with SLEDAl score(r=0.96.P＜0.05),erythrocyte sedimentation rate(r=0.96,P＜0.01),serum level of C reactive protein(r=0.92.P＜0.01)and the titer of antinuclear antibodies(r=0.86,P＜0.01),whereas in 36 patients with leucopenia.a negative correlation was noticed between white blood cell count and the expression level of c-FLIP(r=-0.94,P＜0.0 1).The 23 patients with lupus nephritis had a higher level of c-FLIP than those without lupus nephritis(3.04%±1.09%vs 1.76%±1.09%,t=4.23,P＜0.05).Additionally.the expression of c-FLIP positively correlated with the serum level of IL-4 and IL-10(r=0.80,0.89.respectively,both P＜0.01).Conclusions In patients with active SLE,the expression of c-FLIP is upregulated in peripheral blood B lymphocytes,and positively correlated with the severity of SLE as well as the serum level of IL-4 and IL-10.The upregulation of c-FLIP in B cells might play a certain role in deficient apoptosis or clearance of activated B cells in SLE.

Objective To investigate the expression of glucose transporter type 1(GLUT-1)and hypoxia-inducible factor 1 (HIF-1)α in psoriatic lesions,and to explore their correlations with keratinocyte proliferation.Methods Biopsy specimens were obtained from 30 patients with psoriasis and 20 normal human controls.Immunohistochemistry and Western blotting were used to examine the protein expression of GLUT-1 and HIF-1α in these specimens.Results GLUT-1 and HIF-1α were mainly expressed in the basal layer of the control skin,but throughout the whole epidermis of psoriatic lesions.A significant increase was observed in the expression of GLUT-1 and HIF-1α in psoriatic lesions compared with that in the control skin (botb P＜0.01).In the case of psoriatic lesions,both the expression of GLUT-1 and HIF-1α was positively correlated with that of Ki-67(r=0.70,0.81 respectively,both P＜0.01),and positive correlation was also found between the expression of GLUT-1 and HIF-1α(r=0.85.P＜0.01).Conclusion Our data suggest that uprcgulation Of GLUT-1 and HIF-1α expression in psoriatic lesions might contribute to the proliferation of keratinocytes and psoriasis development.

Objective To construct an eukaryotic expression vector for TAP genes fused with enhanced green fluorescent protein(EGFP)gene,and to analyze the expression and subcellular localization of the fusion protein in A375 human malignant melanoma cells transfected with the eukaryotic expression vector.Methods A375 cells were cotransfected with the combination of plasmid(P)TAP1-EGFP or pTAP2-EGFP and pDsRed2-endoplasmic reticulum(ER),or with pEGFP-TAP1 and-TAP2,or monotransfected with pTAP1-EGFP or pTAP2-EGFP alone.The monoclonal A375 cells stably expressing TAP were obtained by G418 selection.Then.the distribution and expression of fusion proteins were assessed in A375 cells by using fluorescence microscopy and Western blot,respectively.Flow cytometry was used to measure the expression of HLA class Ⅰ on A375 cells.Results Transfection of A375 cells with pTAP1-EGFP or pTAP1-EGFP and pTAP2-EGFP significantly increased the expression of TAP 1 and TAP 2 in as well as HLA class Ⅰ antigen on A375 cells.The green fluorescence of TAP1-EGFP and TAP2-EGFP overlapped with the red fluorescence of ER marker in cotransfected cells.indicating that TAP was localized subcellularly on the ER.Conclusions The expression vector for TAP-EGFP fusion gene has been constructed cuccessfully and expressed in A375 cells,and the expressed fusion protein is subcelluiarly localized to ER.This study will provide a basis for the research into subsequent immune response following induction of TAP expression.

In order to investigate the expression of endothelin receptor B (ETR-B) in human malignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a concentration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
Cell Line, Tumor ; Cell Proliferation/*drug effects ; Endothelin-3/*pharmacology ; Melanoma/*metabolism ; Melanoma/*pathology ; Receptor, Endothelin B/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.

In order to investigate the expression of endothelin receptor B (ETR-B) in human ma-lignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a con- centration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.

Objective: To compare the curative effect of postoperative concurrent chemoradiotherapy (CCRT) and radiation therapy (RT) alone on early-stage cervical cancer patients with intermediate-risk factors. Methods: Clinical data of patients with early stage (ⅠB-ⅡA) cervical cancer who underwent radical hysterectomy and pelvic lymphadenectomy in Zhejiang Cancer Hospital between January 2008 and December 2011 were retrospectively analyzed. Cervical squamous cell carcinoma patients with more than two intermediate-risk factors, including lymphatic vascular infiltration, depth of cervical stromal infiltration >1/2 cm or tumor size >4 cm, were screened. Among them, 80 patients were divided into the RT group, 137 patients were divided into the CCRT group. The survivals of these patients were analyzed. Results: At the end of the follow-up period, a total of 22 patients were dead, one patient was alive with neoplasm. There were 23 patients who occurred recurrence/ metastasis, 14 of them were in the RT group and 9 were in the CCRT group. The 5-year progression free survival (PFS) and 5-year overall survival (OS) rate of the entire cohort were 89.9% and 89.8%, respectively. The 5-year PFS rate of RT group was 82.4%, the 5-year PFS rate of CCRT group was 94.1%, and the difference between the two groups was statistically significant (P=0.013). The 5-year OS rate of RT group was 85.1%, the 5-year OS rate of CCRT group was 92.7%, and the difference between the two groups was statistically significant (P=0.049). Univariate analysis showed that therapeutic modality was associated with the 5-year DFS and OS of early-stage cervical cancer patients with intermediate-risk factors (P<0.05). The results of multivariate analysis showed that therapeutic modality was an independent prognostic factor of favorable PFS (RR=3.741, 95% CI: 1.506-9.289, P=0.004) and OS (RR=2.754, 95%CI=1.143-6.637, P=0.024). Neither of the two groups occurred severe anemia. However, the incidence of mild and moderate anemia in the CCRT group was higher than that of RT group (P<0.001). The incidence of leukocytopenia severer than grade Ⅲ in the CCRT group was significantly higher than that of RT group (P<0.001). However, the patients could recover quickly with the treatment of granulocyte colony-stimulating factor. Conclusion Postoperative CCRT improves the survival of early stage cervical cancer patients with two or more intermediate-risk factors.

Objective:To determine whether postoperative concurrent chemoradiotherapy (CCRT) improves the survival outcomes of cervical cancer patients with pelvic and/or para-aortic lymph node metastasis after radical surgery.Methods:Clinical data of 188 cervical cancer patients presenting with pelvic and/or para-aortic lymph node metastasis after radical surgery between February 2008 and November 2011 were retrospectively analyzed. The incidence of pelvic and/or para-aortic lymph node metastasis was confirmed by postoperative pathology. The clinical efficacy of CCRT was evaluated.Results:Recurrence/metastasis occurred in 46 patients. In the radiotherapy alone group, 4(57.1%) patients had recurrence/metastasis in the posterior peritoneum subgroup, 5(55.6%) in the iliac subgroup and 11(28.2%) in the pelvic non-iliac subgroup, respectively. In the CCRT group, there were 5(62.5%) cases of recurrence/metastasis in the posterior peritoneum subgroup, 5(25%) in the iliac subgroup and 16(15.2%) in the pelvic non-iliac subgroup, respectively. Compared with the radiotherapy alone, CCRT could significantly improve the 5-year overall survival (OS) rate of patients with pelvic without iliac lymph node metastasis or iliac lymph node metastasis (pelvic without iliac: 88.6% vs.76.9%, P=0.003; iliac: 80.0% vs.44.4%, P=0.041), whereas failed to improve the 5-year OS of patients with para-aortic lymph node metastasis (50.0% vs.42.9%, P=0.973). The location of lymph node metastasis and CCRT were the independent prognostic factors for OS (para-aortic vs. pelvic without iliac: hazard ratio[HR]=4.259, 95% CI=1.700-10.671, P=0.002; iliac vs. pelvic without iliac: HR=2.985, 95% CI=1.290-6.907, P=0.011; concurrent chemotherapy vs. radiotherapy alone: HR=0.439, 95% CI=0.218-0.885, P=0.021). Conclusions:CCRT can improve the survival of patients with pelvic lymph node metastasis, but it fails to enhance the survival rate of patients with para-aortic lymph node metastasis.