Curcumin inhibits metastatic progression of breast cancer cell through suppression of urokinase-type plasminogen activator by NF-kappa B signaling pathways.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), is extracted from the plant Curcuma longa. It was recently reported for its anticancer effect on several types of cancer cells in vitro however, the molecular mechanisms of this anticancer effect are not fully understood. In the present study, we evaluated the effects of curcumin on human mammary epithelial carcinoma MCF-7 cells. Cells were treated with curcumin and examined for cell viability by MTT assay. The cells invasion was demonstrated by transwell assay. The binding activity of NF-κB to DNA was examined in nuclear extracts using Trans-AM NF-κB ELISA kit. Western blot was performed to detect the effect of curcumin on the expression of uPA. Our results showed that curcumin dose-dependently inhibited (P < 0.05) the proliferation of MCF-7 cells. Meanwhile, the adhesion and invasion ability of MCF-7 cells were sharply inhibited when treated with different concentrations of curcumin. Curcumin also significantly decreased (P < 0.05) the expression of uPA and NF-κB DNA binding activity, respectively. It is concluded that curcumin inhibits the adhesion and invasion of MCF-7 cells through down-regulating the protein expression of uPA via of NF-κB activation. Accordingly, the therapeutic potential of curcumin for breast cancer deserves further study.

[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.

[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.

[Expression and significance of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase in non-melanoma skin cancer].

OBJECTIVE: To investigate the expression and significance of matrix metalloproteinases (MMP-2, MMP-9) and tissue inhibitor of matrix metalloproteinase (TIMP-2, TIMP-1) in non-melanoma skin cancer (NMSC). METHODS: Thirty six patients with squamous cell carcinoma (SCC) and 32 patients with basal cell carcinoma (BCC), confirmed by pathology, were selected, and 30 cases of normal skin were selected as control. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in all samples were examined by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The expression rate, expression intensity and expression level of each factor were recorded. The results were compared between the groups. RESULTS: The expression rates of MMP-2 and MMP-9 in the control group were 30.0% and 36.7%, the expression levels of MMP-2 and MMP-9 in the control group were 57.216 ± 12.785 and 59.318 ± 13.262, all significantly lower than those in the tumor edge and center of the SCC and BCC groups (P < 0.01). The expression rates of TIMP-1 and TIMP-2 in the control group were 96.7% and 100%, their expression levels were 121.738 ± 25.516 and 122.612 ± 25.964, all significantly higher than those in the SCC and BCC groups (P < 0.01). The expression levels of MMP-2 and MMP-9 in the tumor center and edge of SCC group were significantly higher than those in the corresponding parts of the BCC group, while the expression levels of TIMP-1 and TIMP-2 were significantly lower than those in the BCC group (P < 0.01). The expression levels of MMP-2 and MMP-9 in the tumor edge of the SCC and BCC groups were significantly higher than those in the tumor centers (P < 0.01), while the expression levels of TIMP-1and TIMP-2 were significantly lower than those in the tumor centers (P < 0.01). CONCLUSION: MMP-2, MMP-9 and TIMP-2, TIMP-1 may play an important role in the development, progression, invasion and metastasis of non-melanoma skin cancer.

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.

[Correlation of TOP2A gene expression and survival of breast cancer patients].

OBJECTIVE: The aim of this study was to assess the TOP2A RNA expression and the relationship of TOP2A protein expression with metastasis-free interval in breast cancer patients. METHODS: TOP2A expression was analyzed prior to surgery in 86 patients. The level of TOP2A gene amplification was analyzed by fluorescence in situ hybridization (FISH), its RNA expression level with RT-PCR, and their correlation with TOP2A protein expression was assessed by immunohistochemistry (IHC). The correlation between RNA expression level and metastasis-free interval in breast cancer patients was also analyzed. RESULTS: Aberrations (amplification or deletion) of TOP2A copy number was observed in 25.6% (22/86) of the cases. TOP2A protein expression was detected in 66.3% (57/86) of the samples. There was a significant correlation between the TOP2A RNA expression and protein expression (P < 0.001). TOP2A gene expression was significantly associated with the metastasis-free interval in the breast cancer patients (P = 0.001). There was no significant correlation between TOP2A gene amplification and TOP2A protein expression (P = 0.211). CONCLUSIONS: TOP2A RNA level is an objective and reliable prognostic indicator in breast cancer.

[Expression of IGF-1R in esophageal squamous cell carcinoma and the effect of its silencing by siRNA on the proliferation of esophageal cancer EC9706 cells in vitro].

OBJECTIVE: To explore the correlation of IGF-1R expression with clinical features of esophageal squamous cell carcinoma (ESCC) and to investigate the effect of silencing IGF-1R by siRNA on the proliferation of esophageal cancer cell line EC9706 cells. METHODS: Immunohistochemistry was used to detect the expresion of IGF-1R in 80 specimens of ESCC and 18 specimens of normal esophageal mucosa. IGF-1R siRNA was transfected into esophageal squamous cell carcinoma EC9706 cells, and the effect of RNAi was assessed by Western blot. The proliferation of EC9706 cells was determined by drawing growth curve, MTT assay and plate colony-forming assay. RESULTS: The total and strong positive rates of IGF-1R expression were 86.3% and 51.3% in ESCC, and 61.1% and 11.1% in normal esophageal epithelium, respectively. The total and strong positive rates of IGF-1R expression in patients with lymph node metastasis were 94.4% and 74.1%, significantly higher than 69.2% and 3.9%, respectively, in those without lymph node metastasis (P<0.01). A significantly higher IGF-1R expression was associated with lower histological grade (P<0.05). The total and strong rates of IGF-1R expression in 39 patients of stages III and IV were 97.4% and 71.8% , significantly higher than the 75.6% and 31.7%, respectively, in 41 cases of stages I and II (P<0.01). IGF-1R RNAi significantly inhibited IGF-1R expression and the growth of EC9706 cells. The clone formation rate of RNAi-IGF-1R transfected cells was 19.1%, significantly lower than that of 52.3% in non-transfected cells and 49.0% in empty vector-transfected EC9706 cells (P<0.05). CONCLUSIONS: The overexpression of IGF-1R is colerated with lymph node metastasis, differentiation and clinical stage. Down-regulation of IGF-1R can inhibit the proliferation of esophageal cancer EC9706 cells in vitro.

[Apoptosis and growth arrest of human esophageal squamous cell carcinoma cell EC9706 induced by Fufangkushen injection].

OBJECTIVE: To investigate the inhibitory effect and apoptosis induction on human esophageal carcinoma EC9706 cell by Fufangkushen. METHODS: The experiment of Fufangkushen was designed into three groups including 25.00 µl/ml group, 6.25 µl/ml group and control group in vitro. The method of MTT was used to evaluate the growth inhibition effects. Proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry (IHC) in vitro. The morphological changes of cells were observed under inverted microscope. FACS was used to analyze the distribution of cell cycle and apoptosis. The expressions of Bcl-2, Fas and caspase-3 in EC9706 cells were detected by Western blotting. The clone formation in plate was used to test the capacity of cell clone formation. Nude mice experiments were conducted to investigate the tumor inhibition of Fufangkushen in vivo. The mice were divided into 3 groups of 200 µl/d treatment, 25 µl/d treatment and saline control. PCNA and Bcl-2 were detected by IHC. And the apoptotic index was detected by terminal transferase dUTP nick end labeling (TUNEL) on xenograft of nude mice. RESULTS: The proliferative capacities of 25.00 µl/ml group were lower than that of the control group at 48, 72, 96 h respectively (all P < 0.01). IHC showed the PCNA expressions, cell clone formation rate were both lower than that of control group (in 25.00 µl/ml treatment group both P < 0.05). Many apoptotic cells could be observed. And the apoptotic rate was higher in 25.00 µl/ml group than that in the control group ((25.2 ± 7.3)% vs (3.4 ± 1.5)%, P < 0.01). After a treatment of Fufangkushen, the activation of caspase-3 and the Fas were higher ((21.3 ± 4.4)% vs (1.8 ± 0.6)%, (30.2 ± 8.3)% vs (5.4 ± 1.6)%, both P < 0.01), the Bcl-2 were lower (P < 0.01) were observed in vitro. Comparing with the saline control group, the tumor weight in 200 µl/d treatment group were lower ((987 ± 386) vs (1935 ± 838) mg, P < 0.01) and the apoptotic index higher ((33.8 ± 8.7)% vs (5.3 ± 1.4)%, P < 0.01). CONCLUSION: Fufangkushen can inhibit the proliferation of EC9706 cells and induce the cellular apoptosis. The mechanism of apoptosis is probably associated with the arrest of cell cycle, the up-regulation of Fas, the down-regulation of Bcl-2 and the activation of caspase-3 in ESCC EC9706 cells.

[Insulin induces anticancer cytotoxicity of 5-Fu to two human colon cancer cell lines].

OBJECTIVE: To explore the possibility of use of insulin as a potentiator of 5-Fu to human colon cancer cell lines HCT-8 and HT-29 and study its mechanism. METHODS: MTT assay was used to examine the inhibition rate of cell growth after treatment with 5-Fu and insulin. Cell cycle was determined by flow cytometry. RESULTS: Insulin showed an enhancing effect on the chemotherapeutic response of 5-Fu when insulin was applied at a dose of exceeding 0.8 mU/ml 0 approximately 8 h before 5-Fu. Within the range of from 0.8 mU/ml to 8 mU/ml, a higher concentration of insulin gave a higher proportion of inhibited cells. But when the insulin concentration exceeds 8 mU/ml, the proportion became stable as that of 8 mU/ml. Insulin increased the percentage of S phase cells and decreased the percentage of G(1) phase cells (P < 0.01). The percentage of S phase cells reached a peak when the cells were treated with insulin for 6 hours. CONCLUSION: Insulin can enhance the anticancer toxicity of 5-Fu to human colon cancer cell lines HCT-8 and HT-29 cells. Insulin increases the percentage of S phase cells, which may be one of the main mechanisms of insulin-induced enhancement of anticancer response of cancer cells to 5-Fu chemotherapy.

[Promoting effect of all-trans retinoic acid on the chemosensitivity of esophageal cancer EC9706 cells].

OBJECTIVE: To investigate the impact of all-trans retinoic acid (ATRA) on chemosensitivity to esophageal squamous cell carcinoma EC9706 cells in vitro and its mechanism. METHODS: EC9706 cells were routinely cultured as the control group. The experimental group was divided into three groups. The ATRA group with ATRA in final concentration of 1 µmol/L; the 5-Fu group with 5-Fu in final concentration of 50 mg/L; the combined treatment group with ATRA in final concentration of 1 µmol/L and 5-Fu 50 mg/L. The cell apoptosis was detected by terminal deoxynucleotidy transferase mediated dUTP nick end labelling (TUNEL). The cell cycle and apoptosis were detected by flow cytometry. RESULTS: The results of TUNEL showed that in the combined treatment group appeared a large number of apoptotic cells, and their nuclei were stained brown, with a positive rate of 89.7%. There was a significant difference in the comparison with the ATRA group (38.3%) and 5-Fu group (40.3%) (P < 0.05). The flow cytometry showed that the ATRA + 5-Fu group had a significantly higher apoptosis rate (76.9% ± 2.7%) than that in the ATRA group (38.2% ± 2.6%) and 5-Fu group (45.2% ± 2.3%) (P < 0.05). The ratio of cells in G(1) phase increased in the ATRA + 5-Fu group (83.4% ± 3.0%), significantly higher than (48.2% ± 2.5%) in the ATRA group and (53.2% ± 2.6%) in the 5-Fu group (P < 0.05). The ratio of cells in S + G(2)/M phase was decreased in the ATRA + 5-Fu group, with a significant difference (P < 0.05) when compared with other groups. There was no significant difference between the ATRA group and 5-Fu group (P > 0.05) in the apoptosis rate and the proportion of cells at different phases. CONCLUSION: ATRA can induce apoptosis of esophageal carcinoma EC9706 cells in vitro. The combination of ATRA and 5-Fu may enhance the chemotherapeutic efficacy.

[Mechanism of apoptosis of esophageal cancer EC9706 in nude mice induced by all-trans retinoic acid].

OBJECTIVE: To investigate the mechanism of apoptosis of EC9706 tumor-bearing nude mice induced by all-trans retinoic acid (ATRA). METHODS: Human esophageal carcinoma cell line EC9706 cells were inoculated into nude mice to establish the solid tumor model. The tumor models were divided into the following groups: ATRA group, fluorouracil group, the two-drugs combination group, and with an equal volume fraction of solvent as the control group. The nude mice were sacrificed after 10 days of medication. TUNEL staining was used to detect cell apoptosis. RT-PCR was used to detect the expression level of mRNA and immunohistochemistry was used to detect the expression level of protein of caspase-3 and survivin, the apoptosis-related genes in the tumor tissue. RESULTS: The apoptosis rates of the ATRA group, 5-Fu group and ATRA + 5-Fu group were 44.3%, 39.7% and 91.0%, respectively. There was a significant difference in comparison with the control group (0.7%), and the ATRA group had no significant difference compared with that of the fluorouracil group (P > 0.05), but the apoptosis rate of the two-drugs combination group was significantly higher than that in the two single-drug groups (P < 0.05). The average gray value of caspase-3 protein expressed in the control group was 46.12 ± 0.33 and the relative expression of caspase-3 mRNA was 0.14 ± 0.03, both were significantly lower than that in the ATRA group, 5-Fu group and the two-drugs combination group (P < 0.05). The average gray value of survivin protein expressed in the control group was 96.07 ± 0.13 and the relative expression of survivin mRNA was 0.84 ± 0.04, both were significantly higher than those of other groups (P < 0.05). The ATRA group had no significant difference compared with the fluorouracil group (P > 0.05), but the two-drugs combination group was significantly different compared with the single-drug groups (P < 0.05). CONCLUSION: Apoptosis in the EC9706 tumor cells in nude mice can be induced by ATRA. The mechanism may be related with down-regulation of the level of survivin gene expression and up-regulation of the level of caspase-3 gene expression.

[The relationship between retinoic acid receptor-ß expression and chemotherapy response in esophageal squamous cell carcinoma].

OBJECTIVE: To observe the relationship between expression of retinoic acid receptor-ß(RAR-ß) in esophageal squamous cell carcinoma (ESCC) and chemotherapy response. METHODS: Fifty-two cases advanced ESCC patients treated by DDP and 5-FU, DDP 80 mg/m(2), divided into 5 days; 5-FU 375 mg/m(2), d1-5. Immunohistochemistry was used to examine the expression of RAR-ß in ESCC. Fifty cases normal esophageal tissue were used as controls. RESULTS: RAR-ß immunoreactivity was recognized in both cytoplasm and nucleus, RAR-ß positive rate was lower in ESCC compared with normal tissue (61.5% vs 92%, P < 0.05). The 52 cases ESCC patients were treated 228 chemotherapy cycles, the overall response rate (OR) was 71.2%. The OR in RAR-ß positive patients was 84.4% (27/32), significant higher than RAR-ß negative patients 50.0% (10/20) (P < 0.05). The time-to-progression (TTP) for RAR-ß positive patients was 5.9 months, the median survival period was 12.1 months, 2 years survival rate was 56.7%; whereas TTP for RAR-ß negative patients was 2.1 months, the median survival period was 5.8 months, 2 years survival rate was 32.9%. There was significant difference between the 2 groups (P < 0.05). CONCLUSION: RAR-ß protein expression by immunohistochemistry may be a useful indicator to predict the chemotherapy response and clinical outcome for ESCC, meanwhile it may be an avenue for target therapy.

[Up-regulation of stathmin induces growth arrest of esophageal squamous cell carcinoma EC9706 cell].

OBJECTIVE: To construct a eukaryotic expression vector of stathmin gene and investigate its effect on esophageal squamous cell carcinoma (ESCC) EC9706 cell line. METHODS: Stathmin cDNA coding sequence was amplified by RT-PCR and cloned into a eukaryotic expression vector pcDNA3.1(+). EC9706 cells were transfected with this recombinant plasmid pcDNA3.1-stathmin by lipofectamine. And the stable transfectants were selected with G418 medium. Stathmin protein expression was detected with Western blot in transfected EC9706 cell lines. Morphologic change of stable transfectants was observed under microscope. The proliferation of transfected cells was measured by cell counting, MTT and in vitro formation assay of flat. Flow cytometry was used to detect the cell cycle. Nude mice were adopted to investigate the in vivo tumorigenic characteristics of the transfected cells. RESULTS: A 450 bp coding sequence of stathmin cDNA was amplified by RT-PCR and then cloned into pcDNA3.1(+) plasmid to harvest the recombinant plasmid pcDNA3.1-stathmin. The recombinant plasmid pcDNA3.1-stathmin and blank vector were transfected respectively into EC9706 cells. The up-regulated expression of stathmin protein was validated by Western blot (P < 0.01). Compared with control, EC9706 cells transfected with pcDNA3.1-stathmin appeared swollen and multi-nuclear with a cell mitotic arrest; doubling generation time of pcDNA3.1-stathmin transfectants was prolonged (25 - 28 h); The in vitro cell proliferation ability and clone formation rate (34.5% ± 6.9%) decreased, cell cleavage was blocked at G(2)/M phase (21.7% ± 3.4%) and the oncogenicity of inoculated cells in nude mice decreased (all P < 0.01). CONCLUSIONS: The up-regulated expression of stathmin protein triggered by the recombinant plasmid pcDNA3.1-stathmin can inhibit the proliferation and oncogenicity of ESCC EC9706 cells. This molecule may be a promising therapeutic target in ESCC patients.

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.

[Construction and expression of human stathmin gene eukaryotic expression vector and its effect on esophageal cancer cells].

OBJECTIVE: To construct an eukaryotic expression vector of human stathmin gene and to assess its effect on esophageal cancer EC9706 cells. METHODS: Stathmin cDNA coding sequence was amplified by RT-PCR from Eca109 cells and was cloned into pMD18-T vector. After identifying and sequencing, the correct inserting stathmin gene was sub-cloned into eukaryotic expression vector pEGFP-C2. EC9706 cells were transfected with this recombinant plasmid and control plasmid using Lipofectamine 2000, and the stable intergrant was selected with G418 medium. The expression of enhanced green fluorescent protein (EGFP) protein was detected by fluorescence microscopy and EGFP/stathmin fusion protein by Western blot assay in transfected EC9706 cells. The growth curve of the two stably transfected cells was protracted with cell counting. FACS was used to detect the cell cycle. The clone formation rate in plate and in nude mice was tested to investigate the tumorigenic characteristics of the two stably transfected cells in vitro and vivo. RESULTS: A 450 bp coding sequence of stathmin cDNA was amplified by RT-PCR, which was cloned into pMD18-T vector. After identified with restriction enzyme the recombinant plasmid pMD18-T-stathmin containing reverse inserting sequence was constructed successfully. Then, the sub-clone pEGFP-stathmin was sequenced, confirming that the recombinant vector was right. The recombinant plasmid pEGFP-stathmin and pEGFP-C2 vector were transfected separately into EC9706 cells. After selecting with G418, the cells were transfected steadily. EGFP in EC9706 cells was observed after transfection by fluorescence microscopy. The expressed product was proved to be 46,000 EGFP/stathmin fusion protein by Western blot. Compared with those transfected with pEGFP-C2, the growth of cells transfected with pEGFP-stathmin became slow, the cells were swelled, the cell cycle was blocked at G2/M phase, the average clone formation rate decreased in vitro, and the tumorigenicity of inoculated cells in nude mice was decreased. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-stathmin has been constructed successfully. It expresses steadily in esophageal cancer cells and inhibits the proliferation and tumorigenicity of transfected cells.

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.

[Apoptosis in esophageal cancer cells induced by all-trans retinoic acid].

OBJECTIVE: To study the anti-tumor effects of all-trans retinoic acid (ATRA) and mechanisms of its action. METHODS: Human esophageal carcinoma cell line EC9706 cells were treated with ATRA at different concentration. The proliferation inhibition was examined by MTT assay. Morphological examination, TUNEL method and flow cytometry were used to detect the apoptosis and changes of cell cycle. Immunohistochemical method was used to detect the expression of apoptosis-related genes caspase-3 and bcl-2. The semi-quantification of protein expression was analyzed by pathological image analysis. RESULTS: ATRA inhibited the proliferation of EC9706 cells moderately. Apoptosis in EC9706 cells was induced by ATRA treatment. The morphology of EC9706 cells showed changes such as nuclear chromatin condensation and fragmentation. Sub-G1 peak was found by flow cytometry. The maximal apoptosis rate was 32.6%. The expression of caspase-3 gene was enhanced. The expression of bcl-2 gene was decreased. All these effects were presented in a dose-dependent and time-depend manner. CONCLUSION: Apoptosis is one of the key mechanisms of ATRA action on EC9706 cells.

[Impact of RNA interference targeting hypoxia-inducible factor-1alpha on chemosensitivity in esophageal squamous cell carcinoma cells under hypoxia].

OBJECTIVE: To investigate the impact of RNA interference (RNAi) targeting hypoxia-inducible factor 1alpha (HIF-1 alpha) on chemosensitivity of esophageal squamous cell carcinoma cells under hypoxia. METHODS: Human esophageal squamous cell carcinoma cells of the line EC9706 were cultured and divided into 3 groups: untransfected group, added with cobalt chloride (CoCl(2)), a chemical hypoxia inducer, for 8 h so as to establish a hypoxia model; control siRNA transfected group, transfected with control siRNA, and 30 h after the transfection exposed to CoCl(2) for 8 h; and HIF-1 alpha siRNA-transfected group, transfected with HIF-1 alpha siRNA, and 30 h later exposed to CoCl(2) for 8 h. Western blotting was used to detect the protein expression of HIF-1 alpha. Another EC9706 were cultured and divided into 3 groups to be treated as mentioned above, and then exposed to cisplantin or platixal under normoxic or hypoxic condition. 24 hours later 3-(4, 5-carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay was used to detect the inhibition rates of the cells. Further another EC9706 cells were cultured and then divided into 5 groups: cultured under normoxic condition, cultured under hypoxic condition for 8 h, transfected with control siRNA for 30 h and then under hypoxic condition for 8 h, transfected with HIF-1 alpha siRNA for 30 h and then under hypoxic condition for 8 h. The cell cycle was measured by flow cytometry. RESULTS: The HIF-1alpha protein expression of the HIF-1alpha siRNA group was significantly lower than those of the untransfected and control siRNA transfected groups. The inhibition rates of the EC9706 cells of the groups treated by cisplatin of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.01). The inhibition rates of the EC9706 cells of the groups treated by platixal of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.05) Under hypoxic condition, the inhibition rates of the HIF-1alpha siRNA transfected EC9706 cells treated by cisplatin and platixal of different concentrations were all significantly higher than those of the control siRNA transfected and untransfected EC9706 cells (all P < 0.05). Flow cytometry showed that under hypoxic condition the proportion of cells in G(1)-phase of the EC9706 cells was significantly higher, and the proportion of S-phase cells was significantly lower than those of the normoxic group (both P < 0.05), and under the same hypoxic condition the proportion of the EC9706 cells in G(1)-phase was significantly lower, and the proportion the EC9706 cells in S-phase was significantly higher than those of the normoxic group (all P < 0.05). CONCLUSION: The cell cycle arrest induced by HIF-1alpha may be the mechanism of the resistance to anticancer drugs of the esophageal squamous cell carcinoma cells under hypoxic condition. Blocking HIF-1alpha in esophageal squamous cell carcinoma cells may reverse the multidrug resistance of the tumor cells, so it may offer an avenue for gene therapy.

[Relationship between the expression of hypoxia-inducible factor-1alpha and chemotherapy response in esophageal squamous cell carcinoma].

OBJECTIVE: To observe the relationship between expression of hypoxia-inducible factor (HIF)-1alpha and chemotherapy response and clinical outcome. METHODS: Platixal in combination with cisplatin was used in 48 patients with ESCC at advanced stage. platixal 175 mg/m(2), d1; cisplatin 80 mg/m(2), d 2, d 3, d 4, 21 days was one cycle. Chemotherapy response was evaluated. Specimens mentioned above comes from pathology department, the first affiliated hospital of Zhengzhou university. Immuno-histochemistry was used to examine the expression of HIF-1alpha protein in esophageal squamous cell carcinoma. 20 cases normal tissue of esophago was used as control group. chi(2) test and Kaplan-Merier was used to analysis data. RESULTS: HIF-1alpha immunoreactivity was recognized in both cytoplasm and nuclei. HIF-1alpha protein positive expression rate was 43.75% (21/48) in tumor samples, and was negative expression in normal tissue. Among 21 cases with positive expression of HIF-1alpha, there was none complete response case and 5 cases of partial responses, and the response rate was 23.81%; whereas in 27 cases with negative expression of HIF-1alpha, there were 8 cases of complete response and 11 cases of partial responses, and the response rate was 70.37%. There was significant difference between two groups (P < 0.05). In patients of HIF-1alpha positive expression, the median time to tumor progression was 2.0 months, and the median actuarial survival was 6 months, and survival rate was 13.3% in two years; Whereas in patients of HIF-1alpha negative expression, the median time to tumor progression was 5.0 months, and the median actuarial survival was 11.0 months, and survival rate was 42.2% in two years. There was significant difference between two groups (P < 0.05). CONCLUSION: HIF-1alpha protein expression by immunohistochemistry may be a useful indicator to prediction the chemotherapy response and clinical outcome for esophageal squamous cell carcinoma.

Association between metformin and the risk of gastric cancer in patients with type 2 diabetes mellitus: a meta-analysis of cohort studies.

OBJECTIVES: The objective of this study was to evaluate the association between metformin therapy and the incidence of gastric cancer (GC) in patients with type 2 diabetes mellitus (T2DM). METHODS: We systemically searched the following databases for studies published between the databases' dates of inception and Nov. 2016: PubMed, Embase, the Cochrane Library, the Web of Science, and the China National Knowledge Infrastructure (CNKI). Hazard ratios (HR)and corresponding 95% confidence intervals (CIs) for the association between metformin therapy and the incidence of GC in patients with T2DM were the outcome measures assessed in this study. STATA 12.0 (Stata Corporation, College Station, Texas, USA) was used to conduct the statistical analysis. RESULTS: A total of seven cohort studies including 591,077 patients met all the criteria for inclusion in the analysis. Our data showed that metformin therapy was associated with a significantly lower incidence of GC in patients with T2DM than other types of therapy (HR=0.763, 95% CI: 0.642˜0.905). Subgroup analysis showed that patients living in Taiwan benefitted more from metformin therapy than patients living in any other region, as metformin significantly decreased the risk of GC in patients living in Taiwan but did not significantly decrease the risk of GC in patients living in other regions (HR=0.514, 95% CI: 0.384-0.688). The results of the present analysis support the idea that metformin facilitates reductions in the risk of T2DM-related GC. CONCLUSIONS: The risk of GC among patients with T2DM is lower in patients receiving metformin therapy than in patients not receiving metformin therapy.