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.

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.

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.

[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.

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.

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.

Downregulation of N-cadherin expression inhibits invasiveness, arrests cell cycle and induces cell apoptosis in esophageal squamous cell carcinoma.

Some studies have demonstrated that N-cadherin is upregulated in more invasive cancer cell lines and tumors and plays a key role in intercellular adhesion. However, the understanding on the roles N-cadherin plays in esophageal squamous cell carcinoma (ESCC) is still poor. Our data showed that knock-down of N-cadherin in ESCC cell line (EC9706) could arrest cell cycle at G0/G1 phase, induce cell apoptosis, reduce the invasiveness in vitro, and inhibit the tumor formation in vivo. These results suggest that N-cadherin is an important factor in the progression and metastasis of ESCC and N-cadherin may serve as a potential molecular target for biotherapy of ESCC.

[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.

[Expression of BCSG1-siRNA in tumor transplants of human breast cancer cell line in nude mice].

OBJECTIVE: To investigate the inhibitory effects of siRNA targeting BCSG1 gene expression in tumor transplants of human breast cancer cell line in nude mice. METHODS: Four-pairs of small interfering RNA sequences of BCSG1 were chemically synthesized and inserted into the plasmid expression vectors, and were then transfected into human breast carcinoma cell line MCF7 by liposome method. Plasmid vector with unrelated sequence was used as the vector control. Cells transfected with 4 siRNA sequences, control vector and naive FCF7 cells were transplanted into the nude mice. The tumor inhibition was analysised. Immunohistochemical SP method and semi-quantitative RT-PCR were adopted to detect the BCSG1 mRNA and protein expression, respectively. Breast tissue samples of human infiltrating ductal carcinoma, ductal hyperplasia and fibroadenoma were also used as the controls. RESULTS: The inhibition rates of tumor growth in four BCSG1-siRNA transfected groups were remarkably higher than those of the vector control group and naive MCF7 cells (P<0.01). Compared with that of the vector control and naïve MCF7 cell group, there was a significant decrease of BCSG-1 protein expression in the four experimental groups by immuno-histochemistry staining (P<0.01). In addition, BCSG1 mRNA expression in the four groups transfected with BCSG1-siRNA were significantly less than that of the control vector group, naive MCF7 cell control group and human breast IDC (P<0.01). CONCLUSION: BCSG1-siRNA down-regulates the expression of BCSG1 and inhibits effectively growth of the transplaned human breast cancer cell line in nude mice.

[The effects of ginsenosides Rg3 on the expressions of VEGF and KDR in human lung squamous cancer cells].

OBJECTIVE: To investigate the inhibitive effects of Ginsenosides Rg3 (GS-Rg3) in the process of tumor angiogenesis and the effects on the expressions of VEGF and its receptor KDR in human lung squamous cancer SK-MES-1 cell line. METHODS: Human lung cancer SK-MES-1 cells were cultured in vitro and immunocytochemistry and RT-PCR methods were used to detect the effects of different concentrations of Rg3 on the expressions of VEGF and KDR on SK-MES-1 cells. RESULTS: The immunocytochemistry results showed that the positive rates of VEGF protein in different group of SK-MES-1 cells were 81.33 +/- 9.04, 61.80 +/- 7.98, 43.80 +/- 5.25, 29.77 +/- 8.04, respectively. The positive rates of KDR protein in different group of SK-MES-1 cells were 65.51 +/- 7.45, 51.73 +/- 9.21, 34.87 +/- 6.15, 22.04 +/- 5.11, respectively. There were significant differences between each group. RT-PCR results suggested that with the increase of the concentration of Rg3, VEGF and KDR amplified bands gradually weakened. There were significant differences between each group. CONCLUSION: GS-Rg3 can down-regulate the expressions of KDR and VEGF protein and their mRNA in human lung squamous cancer SK-MES-1 cells. It may be one of the mechanisms in the process of inhibiting tumor angiogenesis.

[Efficacy of Shenyi Capsule combined with gemcitabine plus cisplatin in treatment of advanced esophageal cancer: a randomized controlled trial].

BACKGROUND: In recent years, Shenyi Capsule has been proven to have certain anti-angiogenic effects, and to be effective to many cancers, but its effects on advanced esophageal cancer are scarcely studied. OBJECTIVE: To observe the effects of Shenyi Capsule combined with gemcitabine plus cisplatin (GP) regimen in treatment of advanced esophageal cancer. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Sixty inpatients with advanced esophageal cancer from Henan Tumor Hospital, and the Fist Affiliated Hospital of Zhengzhou University were included and randomly divided into treatment group and control group. There were 30 cases in each group. Patients in the treatment group were treated with Shenyi Capsule combined with GP regimen, and patients in the control group were treated with GP regimen alone. MAIN OUTCOME MEASURES: The total response rate was calculated. The level of vascular endothelial growth factor (VEGF), the chemotherapy side reaction and quality of life in the two groups were evaluated. The follow-up of survival time was conducted too. RESULTS: There was no significant difference in total response rate between the two groups (P=0.264). The levels of VEGF in the two groups were decreased as compared with that before the treatment. After treatment, the VEGF level in the treatment group was lower than that in the control group (P=0.002). The decline rates of white blood cell and blood platelet, and the incidence rate of nausea and vomiting in the treatment group were lower than those in the control group, and there were significant differences between the two groups (P=0.045, P=0.036, P=0.037). The quality of life of the patients in the treatment group was better than that in the control group (P=0.028), and one-year survival rate in the treatment group was higher than that in the control group (P=0.047). CONCLUSION: Shenyi Capsule combined with GP regimen is feasible and safe in treatment of advanced esophageal cancer, and the effects are better than chemotherapy alone. It can improve the total response rate, and is effective in inhibiting new angiogenesis of esophageal cancer, reducing chemotherapy side reaction, and improving the patients' quality of life and survival rates.