[Effects and its mechanism of Nimotuzumab on radiosensitivity of esophageal carcinoma ECA-109 and TE-13 cell lines].

Objective: To investigate the effects of nimotuzumab on radiosensitivity of ECA-109 and TE-13 esophageal carcinoma cell lines and explore its possible mechanism. Methods: The ECA-109 and TE-13 cells were divided into control group, irradiation group, medicine group, and combined group (irradiation + medicine). In the combined group, ECA-109 and TE-13 cells were treated with nimotuzumab for 24 h before irradiation, and the cells were collected 2 h after irradiation. The radiosensitizing effects of nimotuzumab on ECA-109 and TE-13 cells were evaluated by clone formation assay. Cell apoptosis was detected by flow cytometry. Western blotting was used to evaluate the expression of EGFR, p-EGFR, DNA-PKcs, p-DNA-PKcs and γH2AX. Results: The values of Dq (quasithreshold dose), D0(mean lethal dose)and SF2 (surviving fraction at 2 Gy) of ECA-109 and TE-13 cells in the combined group were significantly lower than those of the radiation group (for ECA-109 cells, 1.11 vs. 1.72, 1.40 vs. 2.14, 0.42 vs. 0.66, respectively; for TE-13 cells, 0.41 vs. 0.46, 0.43 vs. 0.65, 0.40 vs. 0.71, respectively (all P<0.05). The sensitivity enhancement ratio (SER) of ECA-109 and TE-13 cells were 1.35 and 1.43, respectively. Flow cytometry showed that the apoptosis rate of ECA-109 and TE-13 cells in the combined group were significantly higher than those of the radiation group [for ECA-109 cells, (41.31±1.52)% vs. (9.54±0.52)%; for TE-13 cells, (46.28±0.28)% vs. (11.32±0.31)%, both P<0.01]. Western blotting showed that the expression levels of EGFR and DNA-PKcs were not significantly different in all groups (all P>0.05). Compared with those of the control group, p-EGFR and p-DNA-PKcs of the radiation group were significantly higher in both cell lines (P<0.05), and the γH2AX levels in the radiation group and medicine group were significantly higher than that of the control group (P<0.05). Compared with those of the radiation group and medicine group, p-EGFR and p-DNA-PKcs protein expression in the combined group were decreased significantly (P<0.05), while γH2AX protein expression was significantly increased (P<0.05). Conclusions: Nimotuzumab can enhance the radiosensitivity of esophageal cancer ECA-109 and TE-13 cells. The potential mechanism may be related to the inhibition of EGFR phosphorylation and down-regulation of DNA damage repair proteins. The radiosensitizing effect of nimotuzumab is greater on poorly differentiated esophageal cancer cells.

[Esophageal adenocarcinoma: the clinicopathologic features, patterns of lymph node metastasis and its influencing factors].

OBJECTIVE: To investigate the clinicopathological characteristics, patterns of lymph node metastasis and the influencing factors in esophageal adenocarcinoma. METHODS: A total of 201 cases of esophageal adenocarcinoma were selected for this study, including 89 cases of pure adenocarcinoma, 57 cases of adenoacanthoma cell carcinoma, 33 cases of mucoepidermoid carcinoma and 22 cases of adenoid cystic carcinoma. A total of 2026 lymph nodes were dissected with an average of 10 lymph nodes. The rule of lymph node metastasis in patients with esophageal adenocarcinoma was analyzed, and the risk factors for lymph node metastasis were identified. RESULTS: Esophageal adenocarcinoma in the middle thoracic esophagus accounted for 50.7% of all patients, and 43.8% in the lower thoracic esophagus. Ninety out of 201 cases (44.8%) had lymph node metastasis. 322 lymph nodes were positive for metastatic adenocarcioma with a metastatic ratio of 15.9% (322/2026). Among the patients with upper-thoracic esophageal carcinoma, 9.1% (1/11) of the cases had lymph node metastasis in the superior mediastinum but no lymph node metastasis was found in the middle mediastinum, lower mediastinal and abdominal lymph nodes. The middle-thoracic esophageal adenocarcinoma showed more extensive lymph node metastasis. Lower mediastinal and abdominal lymph node metastases were common in lower-thoracic esophageal cancer. Multivariate analysis showed that gender, length of lesion, depth of invasion and vascular invasion were independent risk factors for lymph node metastasis in esophageal adenocarcinoma (P=0.010, P=0.006, P=0.000, P=0.019, respectively). Male patients had more lymph node metastasis than female patients (49.1% vs 26.3%,P=0.011). The rates of lymph node metastasis in the tumor length ≤3 cm group, 3.1-5 cm group and >5 cm group were 20.4%, 42.9% and 65.7%, respectively. Lymphatic metastasis rates in the T1, T2, T3, T4 stage cancers were 7.1%, 36.8%, 38.1% and 69.4%, respectively, (P<0.001). Patients with vascular invasion had a higher rate of lymph node metastasis (73.9%) than the patients without vascular invasion (41.0%) (P=0.003). CONCLUSIONS: Most of the esophageal adenocarcinoma are distributed in the middle thoracic esophagus, followed by that in the lower thoracic segment. The lymph node metastasis rate, lymph node metastasis ratio and pattern of lymph node metastasis are similar to those of esophageal squamous cell carcinoma. Male, tumor length, depth of invasion and vascular invasion are risk factors of lymph node metastasis for patients with esophageal adenocarcinoma.

[Patterns and influencing factors of lymph node metastasis of adenocarcinoma of the esophagogastric junction in 393 patients].

OBJECTIVE: To explore the patterns and influencing factors of lymph node metastasis of adenocarcinoma of the esophagogastric junction (AEG). METHODS: Clinicopathological data of 393 AEG patients who underwent radical resection and lymphadenectomy in the thoracic or abdominal cavity were collected. We analyzed the metastatic patterns of 5 119 excised lymph nodes with an average of 13 nodes per patient according to Siewert classification, and the associations between lymphatic metastasis and clinicopathological factors, such as tumor invasion, differentiation, maximum diameter, or pathological type were analyzed. RESULTS: The lymph node metastasis rate and ratio (LNR) were 70.0% (275/393) and 29.1% (1 492/5 119), respectively. All the Siewert subtypes of AEG mainly metastasize downwards to the abdominal lymph nodes, while also spread upwards to the mediastinal lymph nodes. Among them, the lymph node metastasis rate was highest in Siewert type Ⅰ and lowest in Siewert type Ⅲ AEG. The lymph node metastasis rate and ratio in T1, T2, T3, T4 AEGs were 0%, 29.4%, 75.0%, 74.6% and 0%, 10.1%, 14.2%, 32.0%, respectively (χ(2)=35.305, P<0.001 and χ(2)=134.034, P<0.001). The lymph node metastasis rate and ratio of the poorly differentiated adenocarcinoma were 36.0% and 79.3%, respectively, significantly higher than 22.1% and 61.7% of the well-differentiated adenocarcinoma (χ(2)=14.468, P<0.001 and χ(2)=120.009, P<0.001). The lymph node metastasis rate and ratio of patients with a tumor in maximum diameter ≥4 cm were 73.1% and 30.9%, significantly higher than 46.8% and 14.6%, respectively, in the patients with a tumor in maximum diameter of <4 cm (χ(2)=13.636, P<0.001 and χ(2)=64.767, P<0.001). The group of vascular tumor thrombus showed significantly higher lymph node metastasis rate and ratio than those in the group with no vascular tumor thrombus (84.6% versus 67.1%, χ(2)=7.946, P=0.005; and 45.0% versus 26.0%, χ(2)=112.723, P<0.001). The lymph node metastasis ratio of mucinous and signet ring cell adenocarcinoma was 34.9%, significantly higher than 28.5% of the adenocarcinoma (χ(2)=8.710, P<0.001) The depth of tumor invasion and degree of tumor differentiation were independent factors affecting lymph node metastasis (P=0.001 and P<0.001). CONCLUSIONS: The lymph node metastasis rate and ratio of AEG are high and influenced by many clinicopathological factors. The patterns of lymph node metastasis are different among different Siewert subtype AEGs.The depth of tumor invasion and differentiation degree are independent factors affecting lymphatic metastasis.

An antiproliferative gene FLNA regulates migration and invasion of gastric carcinoma cell in vitro and its clinical significance.

This study aimed to analyze the expression and clinical significance of filamin A (FLNA) in gastric carcinoma and the biological effect in its cell line by FLNA overexpression. Immunohistochemistry and western blot were used to analyze FLNA protein expression in 47 cases of gastric cancer and 47 cases of normal tissues to study the relationship between FLNA expression and clinical factors. FLNA lentiviral vector and empty vector were respectively transfected into gastric cancer SGC-7901 cell line. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to detect the mRNA level and protein of FLNA. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and migration and invasion assays were also conducted to determine the influence of the upregulated expression of FLNA that might be found on SGC-7901 cell biological effect. Immunohistochemistry: The level of FLNA protein expression was found to be significantly lower in gastric cancer tissue than normal tissues (P < 0.05). Western blot: The relative amount of FLNA protein in gastric cancer tissue was found to be significantly lower than in normal tissues (P < 0.05). The level of FLNA protein expression was not correlated with gender, age, and tumor invasion (P > 0.05), but it was correlated with lymph node metastasis, clinic stage, and histological grade (P < 0.05). Loss of FLNA expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P < 0.05). The result of biological function showed that SGC-7901 cell transfected FLNA had a lower survival fraction, significant decrease in migration and invasion, and lower matrix metallopeptidase 9 (MMP-9) protein expression compared with SGC-7901 cell untransfected FLNA (P < 0.05). FLNA expression decreased in gastric cancer and correlated significantly with lymph node metastasis, clinic stage, histological grade, and poor overall survival, suggesting that FLNA may play important roles as a negative regulator to gastric cancer SGC-7901 cell by promoting degradation of MMP-9.

Effect of cyclin G2 on proliferative ability of prostate cancer PC-3 cell.

This study aimed to analyze the expression, clinical significance of cyclin G2 (CCNG2) in prostate carcinoma, and the biological effect in its cell line by CCNG2 overexpression. Immunohistochemistry and Western blot were used to analyze CCNG2 protein expression in 85 cases of prostate cancer and normal tissues to study the relationship between CCNG2 expression and clinical factors. CCNG2 lentiviral vector and empty vector were, respectively, transfected into prostate cancer PC-3 cell line. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the mRNA level and protein of CCNG2. MTT assay and cell cycle were also conducted as to the influence of the upregulated expression of CCNG2 that might be found on PC-3 cells biological effect. The level of CCNG2 protein expression was found to be significantly lower in prostate cancer tissue than normal tissues (P < 0.05). The level of CCNG2 protein expression was not correlated with age, PSA contention, and tumor size (P < 0.05), but it was correlated with lymph node metastasis, clinic stage, and Gleason score (P < 0.05). The result of biological function shown that PC-3 cell transfected CCNG2 had a lower survival fraction, more percentage of the G0/G1 phases, and lower CDK2 protein expression compared with PC-3 cell untransfected CCNG2 (P < 0.05). CCNG2 expression decreased in prostate cancer and correlated significantly with lymph node metastasis, clinic stage, and Gleason score, suggesting that CCNG2 may play important roles as a negative regulator to prostate cancer cell.

Expression of BTG1 in hepatocellular carcinoma and its correlation with cell cycles, cell apoptosis, and cell metastasis.

This study aimed to analyze the expression, clinical significance of B cell translocation gene 1 (BTG1) in hepatocellular carcinoma, and the biological effect in its cell line by BTG1 overexpression. Immunohistochemistry and Western blot were used to analyze BTG1 protein expression in 70 cases of hepatocellular cancer and 32 cases of normal tissues to study the relationship between BTG1 expression and clinical factors. Recombinant lentiviral vector was constructed to overexpress BTG1 and then infect hepatocellular cancer HepG2 cell line. The level of BTG1 protein expression was found to be significantly lower in hepatocellular cancer tissue than normal tissues (P < 0.05). Decreased expression of BTG1 was significantly correlated with tumor invasion, lymph node metastasis, clinic stage, and histological grade of patients with hepatocellular cancer (P < 0.05). Meanwhile, loss of BTG1 expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P < 0.05). The result of biological function has shown that HepG2 cell-transfected BTG1 had a lower survival fraction; higher percentage of the G0/G1 phases; higher cell apoptosis; significant decrease in migration and invasion; and lower Cyclin D1 (CND1), B cell lymphoma 2 (Bcl-2), and matrix metalloproteinases (MMP)-9 protein expression compared with HepG2 cell-untransfected BTG1 (P < 0.05). BTG1 expression decreased in hepatocellular cancer and correlated significantly with lymph node metastasis, clinic stage, histological grade, poor overall survival, proliferation, and metastasis in hepatocellular cancer cell by regulating CND1, Bcl-2, and MMP-9 protein expression, suggesting that BTG1 may play important roles as a negative regulator to hepatocellular cancer cell.

Hypoxia suppresses E-cadherin and enhances matrix metalloproteinase-2 expression favoring esophageal carcinoma migration and invasion via hypoxia inducible factor-1 alpha activation.

The goal of this study was to explore the molecular mechanism of hypoxia inducible factor-1 alpha (HIF-1 alpha) action on migration and invasion of esophageal carcinoma cells. We used cobalt chloride (CoCl(2) ) to mimic tumor hypoxic microenvironment and analyzed the expressions of E-cadherin, matrix metalloproteinase-2 (MMP-2), and HIF-1 alpha in esophageal carcinoma cells under hypoxia by reverse transcription polymerase chain reaction and Western blotting. To analyze the function of HIF-1 alpha in Eca109 and TE1 cells, we established stable HIF-1 alpha knockdown cells using small interfering RNA. Blocking effect was detected by Western blotting. The concentrations of MMP-2 protein in the conditioned medium were also determined by enzyme-linked immunosorbent assay. Wound-healing and cell invasion assay were used to evaluate the migration and invasion of esophageal carcinoma cells. After exposure to hypoxia, expressions of HIF-1 alpha protein in Eca109 and TE1 cells were upregulated, both mRNA and protein levels of E-cadherin were downregulated, and MMP-2 were upregulated (P < 0.05), whereas HIF-1 alpha mRNA had no significant change (P > 0.05). Small interfering RNA could block HIF-1 alpha effectively under hypoxia, then enhanced E-cadherin expression and inhibited MMP-2 expression, respectively. Furthermore, expression of HIF-1 alpha protein was stable even though MMP-2 repressed by BB2516. Compared with that in normoxia, Snail expression was enhanced when Eca109 or TE1 cells exposed to hypoxia. Once HIF-1 alpha blocked, Snail expressions were inhibited accordingly. Wound recovery and the number of invading cells decreased (P < 0.05) after HIF-1 alpha blocked. The hypoxia suppresses E-cadherin expression and enhances MMP-2 expression favoring esophageal carcinoma migration and invasion via HIF-1 alpha activation. Our observations suggest that HIF-1 alpha inhibition might be an effective strategy to weaken the migration and invasion of esophageal carcinoma cells.