Blocking OLFM4/HIF-1α axis alleviates hypoxia-induced invasion, epithelial-mesenchymal transition, and chemotherapy resistance in non-small-cell lung cancer.

Hypoxia is a common biological hallmark of solid cancers, which has been proposed to be associated with oncogenesis and chemotherapy resistance. The purpose of the present study was to investigate the role and underlying mechanisms of olfactomedin 4 (OLFM4) in the hypoxia-induced invasion, epithelial-mesenchymal transition (EMT), and chemotherapy resistance of non-small-cell lung cancer (NSCLC). We observed dramatically upregulated expression of OLFM4 in several NSCLC cell lines, and this effect was more pronounced in A549 and H1299 cells. In addition, our data revealed that OLFM4 expression was remarkably increased in both A549 and H1299 cells under hypoxic microenvironment, accompanied by enhanced levels of hypoxia-inducible factor (HIF)-1α protein. The HIF-1α level was elevated in response to hypoxia, resulting in the regulation of OLFM4. Interestingly, OLFM4 was a positive regulator of hypoxia-driven HIF-1α production. Moreover, depletion of OLFM4 modulated multiple EMT-associated proteins, as evidenced by the enhanced E-cadherin levels along with the diminished expression of N-cadherin and vimentin in response to hypoxia, and thus blocked invasion ability of A549 and H1299 cells following exposure to hypoxia. Furthermore, ablation of OLFM4 accelerated the sensitivity of A549 cells to cisplatin under hypoxic conditions, implying that OLFM4 serves as a key regulator in chemotherapeutic resistance under hypoxia. In conclusion, OLFM4/HIF-1α axis might be a potential therapeutic strategy for NSCLC.

Association analysis of genetic variants of adiponectin gene and risk of pancreatic cancer.

Adiponectin is a cytokine exclusively secreted from adipocyte, and could perform direct or indirect effects on anti-inflammation and anti-tumor. Previous researches have studied the correlation between plasma adiponectin levels and the risk of pancreatic cancer. So we aimed at investigating the association of genetic variants of adiponectin gene and the risk of pancreatic cancer. In this study, we genotyped 6 SNPs of adiponectin gene in a case-control study of recruited 172 patients of pancreatic cancer and 181 healthy people in Chinese Han population. The results indicated that two of the SNPs had significant associations with pancreatic cancer. Of which, the SNP rs1501299C>A decreased the risk of PC (P=0.016, OR=0.662 95% CI 0.472-0.928), while rs1065358T>C increased the risk of PC (P=0.027, OR=1.421 95% CI 1.040-1.941). Furthermore, in the clinical correlation analysis, we found rs1501299 was correlated with tumor size (P=0.026), cigarette smoking (P=0.022) and alcohol consumption (P=0.001) and rs1063538 was correlated with alcohol consumption (P=0.026). In conclusion, we provided evidences that the variants in adiponectin gene might influence the development and progression of pancreatic cancer.

TPX2 siRNA regulates growth and invasion of esophageal cancer cells.

PURPOSE: Observe how specific small RNA interference (siRNA) aimed at TPX2 gene suppresses TPX2 gene expression in esophageal cancer EC9706 cells and the effect on esophageal cancer cell growth and invasion ability. METHODS: Transfect TPX2 siRNA into EC9706 cells via lipofectamin 2000. The experiments were divided into three groups, a negative control, a blank control and an siRNA interference group (24h, 48h, 72h, 96h). We examined RNA and protein level alteration of the TPX2 gene after TPX2 siRNA transfection by RT-PCR and Western blot analysis. Detection of how TPX2 siRNA influences EC9706 cell proliferation was done by MTT, cell apoptosis monitored through Tunel assay, in vitro invasion ability via Boyden chamber and cell cycle change by flow cytometry. RESULTS: After effective siRNA transfection, TPX2 mRNA and protein expression level in siRNA interference group were (0.31±0.08, 0.39±0.12),72h after transfection, significantly lower than blank control group (1.00±0.01) and negative control group (0.98±0.11), (F=71.182, t1=8.17, t2=7.90, P<0.05); MTT results demonstrated that cell growth and proliferation were inhibited and the inhibition rate was up to 35.4% (P<0.05) compared with the control group. TUNEL results indicated that cell apoptosis index in siRNA interference group was 18.28±0.35, higher than that in blank control group (4.07±0.26)and negative control group (4.13±0.22), (F=244.5, t1=60.61, t2=53.32, P<0.01). Boyden chamber results showed that the transmembrane cell number was 45.30±8.08 in siRNA interference group, less than blank control group (121.90±7.83), (F=122.46, t1=11.81, t2=10.47, P<0.01); besides, in siRNA interference group cell invasion inhibition rate was 71.42±9.12, higher than negative control group (5.65±3.55), (t=14.256, P<0.01). Flow cytometry results illustrated that more EC9706 cells went into apoptosis and cell cycle arrested in S phase. Similar results were obtained by in vivo transplantation, as TPX2 siRNA transfection significantly reduced tumor growth of the xenograft in nude mice. CONCLUSION: siRNA could effectively inhibit the invasion and metastasis of EC9706 cells, promote the apoptosis of tumor cells and may become a new approach for treatment of esophageal carcinoma.

Epigenetic inactivation of Wnt inhibitory factor-1 in human esophageal squamous cell carcinoma.

Wnt inhibitory factor-1 (WIF1), as one of most important Wnt antagonists, has been detected frequently silenced by promoter hypermethylation in various types of cancer. In this study, we aimed to investigate the promoter methylation profiles of WIF1 in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines, as well as the functional roles of WIF1 in the human ESCC metastatic behavior. WIF1 mRNA levels and promoter methylation status in ESCC tissues and cell lines were detected using RT-PCR and methylation-specific PCR (MS-PCR), respectively. WIF1 protein levels were assessed by Western blot. Stable ESCC cell line with restoration of WIF1 was generated in EC109 cells, which naturally do not express detectable WIF1 mRNA. The effects of reexpressed WIF1 on EC109 cell proliferation and migration were investigated using crystal violet and wound healing assay, respectively. Also the effects of WIF1 reexpression on the beta-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay. WIF1 promoter methylation was frequently observed in ESCC tissues (46%, 23/50) and cell lines (50%, 2/4). Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), increased or restored WIF1 expression in these ESCC cell lines. Restoration of the WIF1 in EC109 cells resulted in a significant inhibition on both cell proliferation and migration. Moreover, reexpression of WIF1 caused significant decrease of beta-catenin/T-cell factor-dependent transcription activity. These findings demonstrated that WIF1 silencing due to promoter hypermethylation is a major mechanism during carcinogenesis of ESCC. This would be an opportunity to prevent the development and progression of HCC through modulation of WIF1.

[Effects of downregulation of HDAC6 expression on cell cycle, proliferation and migration of laryngeal squamous cell carcinoma].

OBJECTIVE: To detect the expression of histone deacetylase 6 (HDAC6) in laryngeal squamous cell carcinoma, and to analyze the effects of downregulation of HDAC6 expression on cell cycle, proliferation and migration of laryngeal squamous cell carcinoma cell line Hep-2 cells, and to explore their possible molecular mechanisms. METHODS: Immunohistochemistry was used to detect the expression of HDAC6 protein in 55 cases of laryngeal squamous cell carcinoma and 20 cases of normal laryngeal mucosa. HDAC6 siRNA and control siRNA were transfected into Hep-2 cells via lipofectamine 2000, and the interfering effect was analyzed using Western blotting. The effects of downregulation of HDAC6 expression on cell cycle, proliferation and migration were determined by cell counting kit-8 (CCK-8), flow cytometry and Boyden chamber, respectively. Finally, Western blotting was used to detect the expressions of cell cycle, proliferation and migration related proteins. RESULTS: There was a high level expression of HDAC6 protein in laryngeal squamous cell carcinoma, and its expression was not related to age and sex of the patients (P > 0.05), but closely associated with the degree of histological differentiation, TNM staging and lymph node metastasis (P < 0.05). HDAC6 siRNA effectively down-regulated the expression of HDAC6 protein in laryngeal squamous cell carcinoma cell line Hep-2 cells, and downregulation of its expression obviously inhibited cell proliferation, arrested cell cycle at G(0)/G(1) phase and decreased cell migration ability in Hep-2 cells. Additionally, the downregulation of HDAC6 protein expression markedly decreased the expressions of cyclin D1, cyclin E, cdk2 and MMP-9 proteins, but increased the expressions of p21 and E-cadherin proteins. CONCLUSIONS: HDAC6 may play a pivotal role in the carcinogenesis and development of laryngeal squamous cell carcinoma. The downregulation of HDAC6 expression-mediated cell proliferation inhibition, cell cycle arrest and decreased cell migration ability may be closely associated with the decrease of cyclin D1, cyclin E, cdk2 and MMP-9 proteins and increase of p21 and E-cadherin proteins.

[Down-regulation of histone deacetylase 2 induces cell apoptosis and inhibits cell proliferation and migration of laryngeal squamous cell carcinoma cells].

OBJECTIVE: To investigate the effects of histone deacetylase 2 (HDAC2) expression on cell proliferation, apoptosis and migration of laryngeal squamous cell carcinoma (LSCC) Hep-2 cells. METHODS: HDAC2 siRNA and control siRNA were transfected into LSCC Hep-2 cells by lipofectamine 2000, and cells were divided into three experimental groups: untreated group, control siRNA group and HDAC2 siRNA transfection group. Western blotting was utilized to detect the expression of HDAC2 protein in Hep-2 cells. Cell proliferation and apoptosis were investigated by CCK-8 kit and flow cytometry, respectively. Boyden chamber was used to study cell migration. Expressions of cell apoptosis and cell migration related proteins were detected by Western blotting. RESULTS: HDAC2 siRNA significantly down-regulated the expression of HDAC2 protein in LSCC Hep-2 cells. Down-regulation of HDAC2 expression coincided with an inhibition of cell proliferation and migration along with an induced cell apoptosis of Hep-2 cells. Moreover, down-regulation of HDAC2 expression significantly increased the expressions of caspase-3 and caspase-9 proteins but decreased the expressions of matrix metalloproteinases (MMP)-2 and MMP-9 proteins. CONCLUSIONS: HDAC2 may play a pivotal role in the initiation and development of LSCC. Down-regulation of HDAC2 expression mediates cell apoptosis. Cell migration inhibition may be tightly associated with overexpression of caspase-3 and caspase-9 along with down-regulation of MMP-2 and MMP-9 expressions.

Downregulation of stathmin is involved in malignant phenotype reversion and cell apoptosis in esophageal squamous cell carcinoma.

BACKGROUND AND OBJECTIVES: Stathmin plays a critical role in the regulation of mitosis and mediates the development of malignant tumors. Here, we investigated the potential role of stathmin in cell cycle and apoptosis in esophageal squamous cell carcinoma (ESCC). METHODS: A stathmin short hairpin RNA (shRNA) plasmid was employed to downregulate stathmin expression in the ESCC cell line EC9706 cells. Cell proliferation was measured by cell counting, MTT, and colony formation assay. Cell migration was measured by Boyden chamber. Western blot was used to analyze the expressions of stathmin, survivin, and apoptosis-related proteins in transfected cells. Cell cycle and apoptosis were determined by flow cytometry and DNA ladder. Oncogenicity assay in nude mice was utilized to analyze phenotypic changes of transfected cells in vivo. RESULTS: After transfection with stathmin shRNA plasmid, stathmin expression markedly decreased in EC9706 cells. Stathmin downregulation significantly inhibited cell proliferation, cell migration in vitro, and tumorigenicity in vivo, meanwhile arrested cell cycle in the G2/M phase and induced cell apoptosis. Further, stathmin downregulation resulted in downregulation of Bcl-2 and survivin proteins, activation of Caspase-3. CONCLUSIONS: These findings demonstrate that stathmin may play an essential role in carcinogenesis of ESCC, which will lay a foundation for target therapy of ESCC.

Overexpression of Pim-1 is associated with poor prognosis in patients with esophageal squamous cell carcinoma.

BACKGROUND AND OBJECTIVES: It is well known that provirus integration site for Moloney murine leukemia virus 1 (Pim-1) plays an essential role in the occurrence and development of tumors. Here, we investigated expression of Pim-1 in esophageal squamous cell carcinoma (ESCC) and assessed the correlation between Pim-1 level and prognosis of the patients with ESCC. METHODS: Expression of Pim-1 was investigated by RT-PCR, immunohistochemistry, and in situ hybridization methods. Kaplan-Meier statistics was used to examine the relationship between levels of Pim-1 and prognosis using log-rank test. RESULTS: The results demonstrated that ESCC tissues appeared strong expression levels of Pim-1 mRNA and protein while the normal esophageal tissues were either negative or showed only weak Pim-1 mRNA and protein levels. In addition, we found that Pim-1 expression was significantly correlated with histology grade, clinical staging, and lymph node metastasis (all P < 0.05), but not related to age and sex (all P > 0.05). Furthermore, the results of survival rates analyzed by Kaplan-Meier curve revealed that patients with high Pim-1 mRNA and protein expressions had a poorer prognosis than those with the low Pim-1 expression (P = 0.010 and 0.001, respectively). CONCLUSION: These findings demonstrate that Pim-1 may be used as molecular marker for predicting the prognosis of patients with ESCC.

[Expression of nucleostemin mRNA and protein in the esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the mRNA and protein expression of nucleostemin (NS) in human esophageal squamous cell carcinoma. METHODS: The mRNA and protein expression of NS were detected in 31 mucosal atypical hyperplasia specimens, 62 esophageal squamous cell carcinoma specimens and the matched normal esophageal mucosa samples by RT-PCR and immunohistochemistry method, respectively. RESULTS: The positive expression rate of NS protein in normal esophageal mucosa, atypical hyperplasia and esophageal squamous cell carcinoma was 17.7% (11/62), 41.9% (13/31) and 69.4% (43/62), respectively. There was a significant difference among the above three groups (chi2 = 33.676, P < 0.01). The expression levels of NS mRNA in esophageal squamous cell carcinoma (0.971 +/- 0.121) was significantly higher than that in the atypical hyperplasia (0.913 +/- 0.085) and also in the normal esophageal mucosa (0.866 +/- 0.103; F = 14.829, P < 0.01). The expression level of both NS protein and mRNA was positively correlated with histological grade, infiltration depth, and lymph node metastasis (P < 0.05), but not with age, gender or pathological type (P > 0.05). CONCLUSION: Our results indicate that nucleostemin mRNA and protein are over-expressed in human esophageal squamous cell carcinoma, and it may be related with its oncogenesis.

[mRNA expression level of nucleostemin in esophageal squamous cell carcinoma tissue].

OBJECTIVE: To investigate the mRNA expression levels of nucleostemin (NS) in human esophageal squamous cell carcinoma tissue. METHODS: Real-time PCR was used to quantify the mRNA expression of NS in the samples of esophageal squamous cell carcinoma tissue and their matched normal esophageal mucosa tissue from 62 patients, 36 males and 26 females, aged (61 +/- 10) (38-75). The relationship between NS mRNA expression level and clinical pathological features was analyzed. RESULTS: The NS mRNA expression level of the 62 cases of esophageal squamous cell carcinoma tissue was(4.5 +/- 2.1), significantly higher than that of the matched normal esophageal mucosa tissue [(2.1 +/- 1.3), t = -5.045, P = 0.000]. The mRNA expression level of NS was associated with tumor grade, depth of infiltration, and lymph node metastasis (all P < 0.05), but not with gender, age, and pathological type (all P > 0.05). Multiple linear regression analysis revealed that clinical and pathological features influenced the NS mRNA expression level (P = 0. 000), and the depth of infiltration and lymph node metastasis were important influencing factors for NS mRNA expression level(both P < 0.05). CONCLUSION: NS may play an important role in the progression and proliferation of esophageal squamous cell carcinoma.

Correlation of matrix metalloproteinase suppressor genes RECK, VEGF, and CD105 with angiogenesis and biological behavior in esophageal squamous cell carcinoma.

AIM: To explore the expression of reversion inducing cysteine-rich protein with Kazal motifs (RECK), vascular endothelial growth factor (VEGF) and endoglin (CD105) protein and its correlation with occurrence, development, invasion and metastasis in esophageal squamous cell carcinoma (ESCC). METHODS: Streptavidin-peroxidase (SP) immunohisto-chemistry was used to detect expression of RECK and VEGF in 62 cases of ESCC, 31 cases of adjacent atypical hyperplastic epithelium and 62 cases of normal esophageal epithelium. CD105 Mb was used to assess microvessel density (MVD). RESULTS: The expression of RECK was closely correlated with histological grade, infiltrative depth and lymphatic metastasis in ESCC (P < 0.05). The expression of RECK decreased during cancer development: normal esophageal epithelium (85.5%, 53/62), adjacent atypical hyperplastic epithelium (71.0%, 22/31), and carcinoma (59.7%, 37/62). There was a significant difference among the groups (P < 0.05). The expression of VEGF protein was closely correlated with infiltrative depth and lymphatic metastasis in ESCC (P < 0.05). The expression of VEGF protein increased during cancer development: normal esophageal epithelium (29.0%, 18/62), adjacent atypical hyperplastic epithelium (54.8%, 17/31), and carcinoma (67.7%, 42/62). There was a significant difference among the groups (P < 0.05). MVDCD105 increased in accordance with histological grade, but there was no significant difference (grade I, 36.92 +/- 10.85; grade II, 37.65 +/- 9.50; and grade III, 38.06 +/- 12.19). The MVDCD105 was closely correlated with infiltration and lymphatic metastasis in ESCC (P < 0.05). The expression of RECK was inversely correlated with the expression of VEGF and CD105. CONCLUSION: RECK, VEGF and CD105 play important roles in the infiltration, metastasis and carcinogenesis in esophageal carcinoma. Angiogenesis in ESCC may be promoted by over-expression of CD105.

[Association of p53, PCNA expression and trace element content in esophageal mucosa].

BACKGROUND & OBJECTIVE: There were abnormal changes of trace element in esophageal cancer patient's hair and serum in the high risk area. But it was still unknown that the relationship between p53 and proliferating cell nuclear antigen(PCNA) expression and the trace element content in varied esophageal mucosa. This study was designed to probe the relationship of trace element content and p53 mutation, PCNA expression in esophgeal mucosa. METHODS: Esophageal biopsy specimen of 151 cases were divided into four groups (normal, esophagitis, dysplasia, and early carcinoma). The quantitative determination of trace element was performed was performed by using X-ray energy spectrometry and the detection of PCNA expression and p53 mutation was performed by using S-P immunohistochemistry method. RESULTS: The contents of Zn, Se, Mo, were 1.74 +/- 0.32, 1.53 +/- 0.64, 0.58 +/- 0.21, 0.20 +/- 0.08; 0.15 +/- 0.06, 0.10 +/- 0.03, 0.04 +/- 0.02, 0; 4.73 +/- 1.31, 3.45 +/- 1.19, 3.51 +/- 1.32, 2.51 +/- 1.04; respectively in four groups. There was a significant difference in varied histological typies(P < 0.05). The contents of Cu/Zn, Ni were 0.57 +/- 0.17, 0.89 +/- 0.18, 2.45 +/- 0.48, 2.92 +/- 1.08; 0.45 +/- 0.05, 1.27 +/- 0.11, 2.46 +/- 0.24, 2.58 +/- 0.33; respectively, which increased gradually in pace with upgrade of esophageal lesions, with significant difference (P < 0.05). The postive rates of p53 and PCNA were 0, 46.15%, 100%; 31.19%, 84.62%, 100% respectively in normal esophageal epithelium, dysplasia, and early carcinoma, with significant difference (P < 0.01), and had significant correlation to trace element. CONCLUSION: The content variation of Zn, Se, Mo, Cu, Ni could be possessed of certain effect on p53 mutation and PCNA overexpression of esophageal epithelium in the high risk area.