Reliability of Chinese web-based ocular surface disease index questionnaire in dry eye patients: a randomized, crossover study.

AIM: To assess the reliability of web-based version of ocular surface disease index in Chinese (C-OSDI) on clinically diagnosed dry eye disease (DE) patients. METHODS: A total of 254 Chinese participants (51% male, 129/254; mean age: 27.90±9.06y) with DED completed paper- and web-based versions of C-OSDI questionnaires in a randomized crossover design. Ophthalmology examination and DED diagnosis were performed prior to the participants being invited to join the study. Participants were randomly designated to either group A (paper-based first and web-based second) or group B (web-based first and paper-based second). Final data analysis included participants that had successfully completed both versions of the C-OSDI. Demographic characteristics, test-retest reliability, and agreement of individual items, subscales, and total score were evaluated with intraclass correlation coefficients (ICC), Spearman rank correlation, Wilcoxon test and Rasch analysis. RESULTS: Reliability indexes were adequate, Pearson correlation was greater than 0.8 and ICCs range was 0.827 to 0.982; total C-OSDI score was not statistically different between the two versions. The values of mean-squares fit statistics were very low compared to 1, indicating that the responses to the items by the model had a high degree of predictability. While comparing the favorability 72% (182/254) of the participants preferred web-based assessment. CONCLUSION: Web-based C-OSDI is reliable in assessing DED and correlation with the paper-based version is significant in all subscales and overall total score. Web-based C-OSDI can be administered to assess individuals with DED as participants predominantly favored online assessment.

Raddeanin A promotes apoptosis and ameliorates 5-fluorouracil resistance in cholangiocarcinoma cells.

BACKGROUND: Bile duct cancer is characterized by fast metastasis and invasion and has been regarded as one of the most aggressive tumors due to the absence of effective diagnosis at an early stage. Therefore, it is in the urgent demand to explore novel diagnostic approaches and therapeutic strategies for bile duct cancer to improve patient survival. Raddeanin A (RA) is extracted from the anemone raddeana regel and has been demonstrated to play antitumor roles in various cancers. AIM: To investigate the effects of RA treatment on bile duct cancer cells. METHODS: In this study, four cholangiocarcinoma cell lines (RBE, LIPF155C, LIPF178C, and LICCF) treated with RA were used to test the cell viability. The RA-associated cell functional analysis, 5-fluorouracil (5-Fu) effectiveness as well as cell cycle- and apoptosis-related protein expression were investigated. RESULTS: RA reduced cell viability in a dose-dependent pattern in four cell lines, and the migration and colony formation abilities were also impaired by RA in RBE and LIPF155C cell lines. RA sensitized cell lines to 5-Fu treatment and enhanced the effects of 5-Fu in cholangiocarcinoma. Also, RA decreased protein expression of Wee1, while the combinational effect of RA and 5-Fu decreased protein expressions of cyclooxygenase-2, B cell lymphoma 2, and Wee1 but increased protein levels of Bax, cyclin D1, and cyclin E. CONCLUSION: Taken together, the results suggest that RA acts as an anti-cancer agent and enhancer of 5-Fu in bile duct cancer cells via regulating multiple cell cycle and apoptosis-related proteins. This finding provides novel clues to exploring a novel antitumor drug for bile duct cancer.

Analysis of cyclin E co-expression genes reveals nuclear transcription factor Y subunit alpha is an oncogene in gastric cancer.

OBJECTIVE: To explore genes potentially co-expressed with cyclin E in gastric cancer and discover possible targets for gastric cancer treatment. METHODS: The Cancer Genome Atlas (TCGA) stomach adenocarcinoma sequencing data were used to predict genes co-expressed with cyclin E. Co-expression genes predicted by cBioPortal online analysis with Pearson correlation coefficient ≥0.4 were analyzed by gene ontology (GO) enrichment annotation using the PANTHER online platform (Ver. 7). Interactions between proteins encoded by these genes were analyzed using the STRING online platform (Ver. 10.5) and Cytoscape software (Ver. 3.5.1). Genes displaying a high degree of connection were analyzed by transcription factor enrichment prediction using FunRich software (Ver. 3). The significant transcription factor and cyclin E expression levels and their impact on gastric cancer progression were analyzed by Western blotting and Kaplan-Meier survival curve analysis. RESULTS: After filtering the co-expression gene prediction results, 78 predicted genes that included 73 protein coding genes and 5 non-coding genes with Pearson correlation coefficient ≥0.4 were selected. The expressions of the genes were considered to be correlated with cyclin E expression. Among the 78 genes co-expressed with cyclin E, 19 genes at the central of the regulatory network associated with cyclin E were discovered. Nuclear transcription factor Y subunit alpha (NF-YA) was identified as a significant transcription factor associated with cyclin E co-expressing genes. Analysis of specimen donors' clinical records revealed that high expression of NF-YA tended to be associated with increased cyclin E expression. The expression of both was associated with progression of gastric cancer. Western blotting results showed that compared with normal tissues, NF-YA and cyclin E were highly expressed in tumor tissues (P < 0.001). Survival curve analysis clearly demonstrated relatively poor overall survival of gastric cancer patients with high cyclin E or high NF-YA expression level, compared to patients with low cyclin E or NF-YA expression (P < 0.05). CONCLUSIONS: NF-YA may promote gastric cancer progression by increasing the transcription of cyclin E and other cell cycle regulatory genes. NF-YA might be a potential therapeutically useful prognostic factor for gastric cancer.

MicroRNA-183 Acts as a Tumor Suppressor in Human Non-Small Cell Lung Cancer by Down-Regulating MTA1.

BACKGROUNDS/AIMS: MicroRNAs (miRs) often contribute to the progression of non-small cell lung cancer (NSCLC) via regulation of mRNAs that are involved in lung homeostasis. We conducted a study aimed at exploring the roles of miR-183 in the proliferation, epithelial-mesenchymal transition (EMT), invasion and migration of human NSCLC cells via targeting MTA1. METHODS: NSCLC and adjacent normal tissues were collected from 194 patients with NSCLC. Positive expression of MTA1 protein was detected by immunohistochemistry. The highest levels of expression of miR-183 were detected using RT-qPCR in SPC-A-1 cells, which were selected and assigned to the following groups: blank, negative control (NC), miR-183 mimic, miR-183 inhibitor, siRNA-MTA1, and miR-183 inhibitor + siRNA-MTA1. The expression of miR-183 and the mRNA and protein expression of MTA1, E-cadherin, Vimentin, Snail, PCNA, Bax and Bcl-2 in tissues and transfected cells were measured using RT-qPCR and western blot analysis. Cell proliferation, apoptosis, migration and invasion were evaluated by CCK-8, flow cytometry, scratch tests and Transwell assays. Tumor xenografts were conducted in nude mice to determine tumor growth. RESULTS: SPC-A-1 cells with the highest levels of miR-183 expression were selected. Compared with adjacent normal tissues, the expression of miR-183 and the mRNA and protein expression of E-cadherin and Bax were decreased in NSCLC tissues, while mRNA and protein expression of MTA1, Vimentin, snail, PCNA and Bcl-2 were increased. MiR-183 was over-expressed in the miR-183 mimic group and under-expressed in the miR-183 inhibitor and miR-183 inhibitor + siRNA-MTA1 groups. In the miR-183 mimic and siRNA-MTA1 groups, the mRNA and protein expression of E-cadherin and Bax, as well as cell apoptosis, were enhanced, while the expression levels of MTA1, Vimentin, snail, PCNA and Bcl-2 mRNA and protein, cell proliferation, migration, invasion and tumor growth were reduced relative to the blank and NC groups. The miR-183 inhibitor group exhibited an opposite trend. CONCLUSION: Our study indicates that miR-183 down-regulates MTA1 to inhibit the proliferation, EMT, migration and invasion of human NSCLC cells.

NDRG2 suppresses proliferation, migration, invasion and epithelial-mesenchymal transition of esophageal cancer cells through regulating the AKT/XIAP signaling pathway.

N-Myc downstream-regulated gene 2 (NDRG2) has recently revealed as a candidate tumor suppressor gene. To inhibit tumor growth and decrease morbidity of esophageal cancer (EC), this study aims to test the hypothesis that the upregulation of NDRG2 may suppress proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of EC cells by regulating the AKT/XIAP signaling pathway. Immunohistochemistry was conducted for the identification of NDRG2, protein kinase B (p-AKT), X-linked inhibitor of apoptosis protein (XIAP) in EC tissues. To identify the regulatory mechanism of NDRG2 on the AKT/XIAP signaling pathway and EMT in EC, over-expressed lentiviral vector and shRNA were applied for up-regulating and interfering NDRG2 expression, and a series of determinations on the biological behavior of EC cells were performed to validate this regulation action. The results of immunohistochemistry showed NDRG2 was lowly expressed in EC tissues while p-AKT and XIAP are highly expressed. Over-expression of NDRG2 suppresses the proteins related to AKT/XIAP signaling pathway and EMT. Besides, a series of determinations shows the proliferation, migration and invasion of TE-13 cells were suppressed by over-expressed NDRG2, while the cell cycle progression was blocked and cell apoptosis was promoted. And in vivo experiment also demonstrated NDRG2 could inhibit tumor growth. Our findings demonstrate over-expression of NDRG2 works as tumor suppressive role in EC through its effects on inhibition of cell migration, invasion, and EMT by inhibiting the AKT/XIAP signaling pathway.

Licochalcone A inhibits PI3K/Akt/mTOR signaling pathway activation and promotes autophagy in breast cancer cells.

Previous studies have demonstrated that Licochalcone A possesses anti-inflammatory, anticancer, anti-bacterial, anti-malarial and anti-parasitic activities. In the present study the potential anticancer effects of Licochalcone A on MCF-7 cells were investigated. Licochalcone A significantly decreased cell viability and promoted autophagy and apoptosis, as demonstrated by an MTT assay, acridine orange staining and Annexin V-fluorescein isothiocyanate staining, respectively. Western blot analyses demonstrated that Licochalcone A treatment activated the LC3-II signaling pathway while suppressing the phosphoinositide 3-kinase (PI3K)/RAC-α serine-threonine-protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. In addition, Licochalcone A significantly increased caspase-3 activity and significantly decreased B-cell lymphoma-2 expression. The results from the present study indicate that Licochalcone A inhibits PI3K/Akt/mTOR activation, and promotes autophagy and apoptosis in MCF-7 cells.

Expression of P-EGFR and P-Akt protein in esophageal squamous cell carcinoma and its prognosis.

The phosphorylated epidermal growth factor receptor (P-EGFR) and phosphorylated Akt (P-Akt) protein in esophageal squamous cell carcinoma (ESCC) were studied, and its significance in clinical prognosis of patients was assessed. The expression of P-EGFR and P-Akt protein in 83 cases of ESCC and 83 normal esophageal tissues was determined by immunohistochemical staining. Log-rank test and correlation analysis were used to analyze the prognosis of ESCC. The positive expression of P-EGFR in ESCC was 88% (73/83 cases) compared with 41% in normal esophageal mucosa (34/83 cases) (P<0.05). The rate of P-Akt protein expression in ESCC was 90.4% (75/83 cases), compared with 27.7% seen in normal esophageal mucosa (23/83 cases) (P<0.05). The expression of P-EGFR and P-Akt protein was positively correlated with lymph node metastasis and degree of differentiation (P<0.05) irrespective of sex, age, tumor diameter and TNM stage (P>0.05). The expression of P-EGFR was positively correlated with that of P-Akt protein (r=0.674, P<0.01). P-EGFR expression was negatively correlated with survival time of patients with ESCC (r=-0.526, P<0.01). The Kaplan-Meier survival curves showed that the cumulative survival rate of P-EGFR-positive cases was significantly lower than that of the P-EGFR-negative cases (P<0.01). The expression of P-Akt was negatively correlated with survival in patients with ESCC (r=-0.473, P<0.01). The Kaplan-Meier survival curves showed that the cumulative survival rate of the P-Akt-positive cases was significantly lower than that of the P-Akt-negative cases (P<0.01). In conclusion, P-EGFR and P-Akt protein expression is closely related to the incidence of ESCC and mediates the development of invasive cancer and metastasis. It is used to determine the prognosis of ESCC, and may represent a new therapeutic target for the disease.

P-mTOR Expression and Implication in Breast Carcinoma: A Systematic Review and Meta-Analysis.

OBJECTIVE: Phosphorylated mammalian target of rapamycin (p-mTOR) is a promising prognostic marker in many types of cancer. However, its survival benefit in patients with breast carcinoma remains unknown. The aim of the present study was to assess the relationship between p-mTOR expression and prognosis in breast carcinoma based on a systematic review and meta-analysis. MATERIALS AND METHODS: Electronic databases (including Pubmed, Embase, ISI web of science, and Cochrane Library) were searched up to November 24, 2015. The outcome measures were hazard ratios (HRs) with 95% confidence interval (CI) for the association between the prognosis of breast carcinoma patients and p-mTOR expression. Primary end points were disease-free survival (DFS), overall survival (OS), and recurrence-free survival (RFS). Statistical analysis was performed with STATA 12.0. RESULTS: Nine cohort studies including 3051 patients met full eligibility criteria. The pooled HRs (95% CI) for OS, DFS, and RFS were 0.84 (0.27-2.63), 0.71 (0.40-1.23), and 0.48 (0.20-1.18), respectively. CONCLUSIONS: Our findings suggested that p-mTOR overexpression was not significantly related to prognosis in breast carcinoma regarding OS and disease recurrence. Prospective studies are warranted to examine the association between p-mTOR expression and survival outcomes in breast carcinoma.

Icotinib versus docetaxel used in lung adenocarcinoma patients who failed platinum-based chemotherapy: a retrospective study.

BACKGROUND: The efficacy and safety of epidermal growth factor receptor tyrosine kinase inhibitors have been studied worldwide. However, there are few reports directly comparing the efficacy and safety between icotinib and docetaxel as second-line treatment in lung adenocarcinoma patients who have failed platinum-based chemotherapy. This article offers insight into this field. METHODS: A total of 137 patients with stage III or IV lung adenocarcinoma who had progressed on first-line platinum-based therapies and received icotinib or docetaxel therapy between October 2011 and February 2013 were retrospectively reviewed. Patients in the icotinib group received oral icotinib at a dose of 125 mg tid, while patients in the docetaxel group received infusion docetaxel at a dose of 75 mg/m(2) on day 1 of every 21 days (four to six cycles) until disease progression or unacceptable toxicity occurred after which best supportive care was given. RESULTS: There was no statistically significant difference in the objective response rate (23.3% vs 12.5%, P=0.103), progression-free survival (121 days vs 106 days, P=0.083), and overall survival (307 days vs 254 days, P=0.070) between the two groups. As compared to the docetaxel group, the disease control rate (75.3% vs 54.7%, P=0.011) was significantly better in the icotinib group. In the icotinib group, the most common adverse events were rash (35.62%) and diarrhea (24.66%), whereas in the docetaxel group, elevation of transaminase (37.50%), leukopenia (50.00%), and anemia (54.69%) were the most common. CONCLUSION: Icotinib had similar efficacy and a lower adverse events rate in epidermal growth factor receptor-unselected patients as compared to docetaxel, thereby making it an effective second-line therapy option for lung adenocarcinoma.

PU.1 Is Identified as a Novel Metastasis Suppressor in Hepatocellular Carcinoma Regulating the miR-615-5p/IGF2 Axis.

Invasion and metastasis is the major cause of tumor recurrence, difficulty for cure and low survival rate. Excavating key transcription factors, which can regulate tumor invasion and metastasis, are crucial to the development of therapeutic strategies for cancers. PU.1 is a master hematopoietic transcription factor and a vital regulator in life. Here, we report that, compared to adjacent non-cancerous tissues, expression of PU.1 mRNA in metastatic hepatocellular carcinoma (HCC), but not primary HCC, was significantly down-regulated. In addition, levels of PU.1 mRNA in metastatic hepatoma cell lines MHCC97L and MHCC97H were much lower than in non-metastatic Hep3B cells. Transwell invasion assays after PU.1 siRNA transfection showed that the invasion of hepatoma cell lines was increased markedly by PU.1 knockdown. Oppositely, overexpression of PU.1 suppressed the invasion of these cells. However, knockdown and overexpression of PU.1 did not influence proliferation. Finally, we tried to explore the potential mechanism of PU.1 suppressing hepatoma cell invasion. ChIP-qPCR analysis showed that PU.1 exhibited a high binding capacity with miR-615-5p promoter sequence. Overexpression of PU.1 caused a dramatic increase of pri-, pre- and mature miR-615-5p, as well as a marked decrease of miR-615-5p target gene IGF2. These data indicate that PU.1 inhibits invasion of human HCC through promoting miR-615-5p and suppressing IGF2. These findings improve our understanding of PU.1 regulatory roles and provided a potential target for metastatic HCC diagnosis and therapy.

N-cadherin participated in invasion and metastasis of human esophageal squamous cell carcinoma via taking part in the formation of vasculogenic mimicry.

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks, and the presence of VM correlates to an increased risk of metastasis and poor clinical outcome of cancers. Several key molecules, including N-cadherin, have been implicated in VM. However, the role of N-cadherin in the formation of VM in esophageal squamous cell carcinoma (ESCC) had not been elucidated. In this study, firstly we aimed to identify VM patterns in ESCC tissues and to explore their clinical significance. VM was present in 12 out of 56 samples, and ESCC with lymph node metastasis had a higher incidence of VM than that without lymph node metastasis. More importantly, VM channels were associated with the expression of N-cadherin in ESCC tissues. In order to further explore the role of N-cadherin in VM formation and invasion and metastasis in ESCC, secondly, we silenced the expression of N-cadherin with small hairpin RNA in ESCC cell line KYSE-70; herein, we showed that KYSE-70 cells with N-cadherin silencing lost not only the capacity to form tube-like structures on collagen (VM) but also the invasion, metastasis and proliferation ability in KYSE-70 cells in vitro. Taken together, antivascular therapies targeting tumor cell VM may be an effective approach to the treatment of patients with highly metastatic ESCC.

MYC and MXI1 protein expression: potential prognostic significance in women with breast cancer in China.

OBJECTIVE: To investigate the expression levels and the clinical significance of MYC and MXI1 proteins in breast cancer. METHODS: The expression levels of MYC and MXI1 were detected by immunohistochemical assay in 166 cases of breast cancer; the relationships among MYC, MXI1 and the clinicopathological parameters were analyzed by χ2 test. Univariate analysis and Cox's proportional hazards model were used to evaluate the prognostic significance of the 2 proteins. RESULTS: 27.71% of the tumor specimens showed high staining intensity for MYC (high-expression group, HEG-MYC) and 22.89% showed high staining intensity for MXI1 (HEG-MXI1); the expression of 2 proteins was negatively correlated (r = -0.177 p = 0.022). The Kaplan-Meier method for survival analysis showed that patients of the MYC-HEG demonstrated a significantly worse disease-specific survival than those of the MYC-low-expression group (LEG) (χ2 = 11.102, p = 0.001). However, patients of the MXI1-HEG had a significantly better disease-specific survival than those of the MXI1-LEG (χ2 = 7.858, p = 0.005). Both univariate analysis and Cox's proportional hazards model indicated that MYC and MXI1 could be independent prognostic molecular markers. CONCLUSION: MYC-HEG and MXI1-LEG levels are associated with poor prognosis in patients with breast cancer, suggesting that they may be useful molecular markers in breast cancer prognosis prediction.

Tamoxifen-resistant breast cancer cells possess cancer stem-like cell properties.

BACKGROUND: Cancer stem cells (CSCs) are the cause of cancer recurrence because they are resistant to conventional therapy and contribute to cancer growth and metastasis. Endocrinotherapy is the most common breast cancer therapy and acquired tamoxifen (TAM) resistance is the main reason for endocrinotherapy failure during such therapy. Although acquired resistance to endocrine treatment has been extensively studied, the underlying mechanisms are unclear. We hypothesized that breast CSCs played an important role in TAM-induced resistance during breast cancer therapy. Therefore, we investigated the biological characteristics of TAM-resistant (TAM-R) breast cancer cells. METHODS: Mammosphere formation and tumorigenicity of wild-type (WT) and TAM-R MCF7 cells were tested by a mammosphere assay and mouse tumor xenografts respectively. Stem-cell markers (SOX-2, OCT-4, and CD133) and epithelial-mesenchymal transition (EMT) markers were tested by quantitative real-time (qRT)-PCR. Morphological observation was performed to characterize EMT. RESULTS: After induction of TAM resistance, TAM-R MCF7 cells exhibited increased proliferation in the presence of TAM compared to that of WT MCF7 cells (P < 0.05), indicating enhanced TAM resistance of TAM-R MCF7 cells compared to that of WT MCF7 cells. TAM-R MCF7 cells showed enhanced mammosphere formation and tumorigenicity in nude mice compared to that of WT MCF7 cells (P < 0.01), demonstrating the elevated CSC properties of TAM-R MCF7 cells. Consistently, qRT-PCR revealed that TAM-R MCF7 cells expressed increased mRNA levels of stem cell markers including SOX-2, OCT-4, and CD133, compared to those of WT MCF7 cells (P < 0.05). Morphologically, TAM-R MCF7 cells showed a fibroblastic phenotype, but WT MCF7 cells were epithelial-like. After induction of TAM resistance, qRT-PCR indicated that MCF7 cells expressed increased mRNA levels of Snail, vimentin, and N-cadherin and decreased levels of E-cadherin, which are considered as EMT characteristics (P < 0.05). CONCLUSION: TAM-R MCF7 cells possess CSC characteristics and may be responsible for TAM resistance during breast cancer therapy.

Chemoradiotherapy followed by surgery could improve the efficacy of treatments in patients with resectable esophageal carcinoma.

BACKGROUND: The effectiveness of chemoradiotherapy followed by surgery (CRTS) in patients with resectable esophageal carcinoma remains controversial. We performed a systematic review of the literature with meta-analysis. METHODS: Electronic databases were used to identify published studies between January 1992 and April 2012. Pooled relative risk (RR) with 95% confidence interval (95% CI) was utilized to estimate the strength of the association between CRTS and surgery alone (SA) survival of the resectable esophageal carcinoma patients. Heterogeneity and publication bias were also assessed in the present study. RESULTS: The final analysis of 2755 resectable esophageal carcinoma cases from 21 randomized controlled trials (RCTs) are presented. Compared to the SA group, the 1, 3- and 5-year survival rates were significantly higher in the CRTS group (all P < 0.05); the 3- and 5-year survival rates for the Eastern patients, Western patients, patients undergoing concurrent chemoradiotherapy, patients with squamous cell carcinoma, patients undergoing High-dose radiotherapy (≥ 40 Gy), and patients given either "cisplatin + Fluorouracil" or "cisplatin + paclitaxel" chemotherapy were significantly higher in the CRTS group (all P < 0.05). There were no statistical significances in the 3- and 5-year survival rates for patients undergoing sequential chemoradiotherapy or patients with adenocarcinoma between the two groups (all P > 0.05). Compared to the RCTS group, the surgery rate in the SA group was higher (P < 0.05), while the CRTS group had significantly higher radical resection rate, R0 resection rate and lower postoperative local recurrence rate (all P < 0.05). The differences in postoperative complication incidence, post-operative distant metastasis and postoperative mortality rate were not statistically significant between the two groups (all P > 0.05). CONCLUSION: CRTS can significantly improve the survival and surgical conditions of patients with resectable esophageal carcinoma.

HDAC inhibitor trichostatin A suppresses esophageal squamous cell carcinoma metastasis through HADC2 reduced MMP-2/9.

PURPOSE: The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has been shown to act as an anti-tumor agent; however, the effect and mechanism of TSA on the invasion of esophageal squamous cell carcinoma (ESCC) remains unknown. METHODS: To determine whether TSA suppresses the invasiveness of ESCC cell via HDAC2, the expression of HDAC2 in ESCC tissues and adjacent non-tumor tissues were compared using Western blot and immunohistochemistry. Cells were transfected with HDAC2 siRNAs and non-targeting control siRNA using Lipofectamine TM 2000. Cell invasion was investigated using a transwell assay. The protein levels of matrix metalloproteinase-2/9 (MMP-2/9) were examined by Western blot analysis. RESULTS: Expression of HDAC2 was significantly higher in ESCC than in adjacent non-tumor tissues. Additionally, the in vitro invasion assay found that both downregulation of HDAC2 expression and TSA treatment inhibited ESCC cell invasion by approximately 75%. Also, an MMP2/9-specific inhibitor sharply suppressed ESCC cell invasion. Furthermore, both downregulation of HDAC2 and treatment with TSA decreased MMP-2 and MMP-9 protein levels in ESCC cells. CONCLUSIONS: These results suggest that the inhibitory effect of TSA on cancer invasion is mediated through the suppression of HDAC2 expression, and that the reduction of MMP-2 and MMP-9 expression induced by HDAC2 may be involved in the anti-invasive effect of TSA.

Synuclein-γ suppression mediated by RNA interference inhibits the clonogenicity and invasiveness of MCF-7 cells.

The aim of the present study was to investigate the effects of synuclein-γ (SNCG) downregulation by RNA interference (RNAi) on the clonogenicity and invasiveness of MCF-7 breast cancer cells. This study used four pairs of SNCG-specific siRNAs which were designed and cloned into the pGPU6 plasmid for introduction into an MCF-7 cell line. The SNCG knockdown efficacies of the four siRNAs were compared using the reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. The cells' clonogenic and invasive phenotypes were examined with clonogenic and Boyden chamber assays. In comparison with the non-specific siRNA and empty vector controls, all four SNCG siRNAs were observed to significantly inhibit SNCG expression at the mRNA and protein levels (F=481.06, P<0.001; F=147.42, P<0.0001). SNCG suppression mediated by RNAi successfully inhibited the clonogenicity (P=0.002) and invasiveness (P<0.001) of transfected MCF-7 cells. According to the results of the present study, we concluded that SNCG suppression mediated by RNAi significantly suppressed SNCG expression at the mRNA and protein levels, suggesting that SNCG suppression mediated by an RNAi strategy may become a novel approach for treating advanced breast cancer.

[Arsenic trioxide restores ERα expression in ERα-negative human breast cancer cells and its treatment efficacy in combination with tamoxifen in xenografts in nude mice].

OBJECTIVE: To study the demethylation effect of arsenic trioxide (As2O3) on ERα-negative human breast cancer MDA-MB-435s cells and its possible mechanisms, and to observe its treatment efficacy in combination with tamoxifen (TAM) after ERα re-expression. METHODS: MTT assay was used to examine the inhibitory effect of As2O3 treatment alone or in combination with TAM on cell proliferation. A nude mouse xenograft model was used to further examine the treatment efficacy in vivo. MSP was used to detect the methylation status of ERα gene after treated with As2O3 in MDA-MB-435s cells and the transplanted tumor tissues. RT-PCR was used to detect the mRNA expression of DNMT1 and Erα. Western bolt was used to detect the DNMT1 and ERα protein expression. The diameter of xenograft tumors was measured weekly, and the tumor growth curve was drawn. RESULTS: The level of proliferation of the MDA-MB-435s cells was significantly suppressed after treatment with different concentration of As2O3 alone or As2O3 combined with TAM, and the 4 µmol/L As2O3 + TAM treatment for 72 h showed the highest inhibition rate (62.6%). 1, 2, 4 µmol/L As2O3 had demethylation effect on MDA-MB-435s cells, and the DNMT1 mRNA and protein expression was inhibited and accompanied by ERα mRNA and protein re-expression. The unmethylation specific bands of ERα gene were enhanced after treated by As2O3 alone or As2O3 combined with TAM in the xenograft tumors. The expression of DNMT1 mRNA and protein was inhibited, and accompanied by ERα mRNA and protein re-expression. An significant decrease of volume and weight of the xenograft tumors in the As2O3 treated alone or combined with TAM groups was observed compared with those of the normal saline group or TAM alone group (P < 0.05), and the 4 mg/kg As2O3 + TAM group had the highest inhibition rate of tumor weight (79.5%) and volume (76.4%). CONCLUSIONS: ERα can be re-expressed in ERα-negative breast cancer MDA-MB-435s cells after treated with As2O3 by inhibiting the DNMT1 activity. MDA-MB-435s cells are re-sensitized to endocrine therapy after ERα re-expression. As2O3 combined with TAM may provide a new therapeutic approach for patients with ERα-negative breast cancer in the clinic.

[Small interfering RNA in silencing cox-2 expression enhances radiosensitivity of esophageal cancer EC9706 cell].

OBJECTIVE: To explore the effects of small interfering RNA (siRNA) specific to cox-2 gene on the radiosensitivity of esophageal cancer cell EC9706. METHODS: The siRNA vector was established for cox-2 gene and then induced into esophageal cancer cell EC9706 by lipofectamine. G418 screening yielded stably transfected cells. After the irradiation of 0, 2 and 4 Gy, the cellular expression levels of cox-2, matrix metalloproteinase-2 (MMP2), Bax and Bcl-2 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and those of cox-2 protein, AKT protein and phosphorylation AKT protein (pAKT) by Western blot. Cell apoptosis was examined by flow cytometer. Invasion of cells was detected by invasive assay in vitro. The invasive and metastatic capacities of cancer cells were assessed by invasion assay in vitro. Proliferative potential was quantified by clone-forming assay. RESULTS: The sequencing result confirmed that siRNA vector pRNA-U6 for cox-2 gene was established. The results of 1-sinCox214, RT-PCR and Western blot showed that cox-2 gene expression of transfected EC9706 cell was silenced efficiently. After the irradiation of 0, 2 and 4 Gy, the expressions of MMP2, Bcl-2 mRNA, AKT protein and pAKT in silencing cox-2 gene expression significantly decreased. There was an inverse correlation with irradiation dose. The Bax mRNA expression evidently increased directly with irradiation dose; the apoptotic rate in cox-2 silencing groups was evidently higher than the control groups. And the difference was significant (P < 0.01); invasion cells in vitro in Cox-2 silencing groups evidently decreased with significant difference (P < 0.01). The colony formation rate of cells decreased obviously in cox-2 silencing groups after the irradiation of 0, 1, 2, 4, 6, 8 and 10 Gy (P < 0.01). CONCLUSIONS: Small interference RNA in silencing cox-2 gene expression can enhance significantly the radiosensitivity of esophageal cancer EC9706 cells. And the mechanism may be related with MMP2, Bax, Bcl-2, AKT protein and pAKT protein.

[Roles and mechanisms of E2F-1 and PAC1 in signaling oxidative stress-induced apoptosis of Saos-2 cells].

OBJECTIVE: To explore the roles and mechanisms of E2F-1 and PAC1 in signaling apoptosis of Saos-2 cells under oxidative stress. METHODS: siRNAs were used to construct the cell clones of expressing siE2F-1 and siPAC1. E2F-1/PAC1-mediated induction of apoptotic cell death in response to H2O2 were examined by trypan blue exclusion and the expression levels of related target factors detected by Western blot. RESULTS: The cell viabilities of Saos-2/siE2F-1 and Saos-2/siPAC1 increased markedly comparing with the control cells after the treatment of H2O2. And the expression level of p-ERK1/2 was higher than that of the control cells. CONCLUSIONS: The pathway of E2F-1/PAC1/MAPKase is a specific cascade for apoptotic signaling.

Increased expression and function of P-glycoprotein in peripheral blood CD56+ cells is associated with the chemoresistance of non-small-cell lung cancer.

PURPOSE: Chemoresistance is common among non-small-cell lung cancer (NSCLC), P-glycoprotein (P-gp), encoded by the human multi-drug-resistant MDR1 gene, and multidrug-resistance protein 1 (MRP1) might be major contributors. The aim of the present study was to develop an effective method to investigate the expression and function of P-gp in the peripheral CD56+ cells in order to clarify their correlation with the chemoresistance in NSCLC. METHODS: Using microbead technology and a RT-qPCR methodology, we evaluated the expression levels of P-gp and MRP1 in the purified CD56+ cells in the chemoresistance and chemo-naive NSCLC patients compared with that in the healthy volunteers. Flow cytometric analysis was used to investigate the changes of P-gp function in the CD56+ cells between the three cohorts. RESULTS: The MDR1 gene expression was elevated markedly (twofold-tenfold), and P-gp function was increased in the chemoresistance cohort compared with the chemo-naive and the healthy cohorts; whereas there was only about two times averagely elevated for the MRP1 gene expression. No statistical significance (p > 0.05) was seen with respect to the expression of MDR1 and MRP1, the function of P-gp between the chemo-naive and the healthy cohorts. CONCLUSIONS: P-gp in peripheral CD56+ cells demonstrated possible clinical relevance as predictive biomarkers for the identification of chemoresistance in NSCLC, while MRP1 may not play a significant role in the drug resistance in NSCLC. The potential applications for this finding are provided evidence to screen the potential P-gp reversors and to diagnose and manage the chemoresistance in NSCLC patients.