Positive expression of chemokine (C-C Motif) ligand 18 and prognosis in cancer: A meta-analysis.

PURPOSE: Chemokine (C-C Motif) Ligand 18 (CCL18) is a chemotactic cytokine involved in the pathogenesis and progression of various cancers by activating downstream signaling pathways and affecting cellular behaviors. We conducted a meta-analysis to evaluate the CCL18 as a prognostic marker for cancer and determine the relationship between CCL18 and clinicopathological features of cancer. METHODS: We searched the PubMed, Cochrane, Embase, Web of Science and SinoMed databases for publications to investigate the association between CCL18 expression and survival outcome in cancer. Hazard ratios (HRs) and 95% confidence intervals (CI) of overall survival (OS) were pooled. Odds ratios (ORs) of clinicopathological features were computed. Meta-analysis was performed using STATA 12.0 software. RESULTS: Our meta-analysis identified a total of 17 studies including 2829 cases. Meta-analysis revealed that the expression of CCL18 in various cancer tissues was significantly higher than that in the normal group (OR=16.694, 95% CI=14.117-27.476, p<0.01, random effects). The abnormal expression of CCL18 was associated with lymph node metastasis (OR=4.409, 95% CI=2.129-9.128, p<0.01, random effects) and TNM stage (breast cancer subgroup: III+IV vs I+II OR=13.187, 95% CI=8.417-20.660, p<0.01; gastric cancer subgroup: III+IV vs I+II OR=0.034, 95% CI=0.008-0.137, p<0.01) but is was not related to gender (male vs. female: OR=0.88, 95% CI=0.667-1.162, p=0.368) and age (>60 vs. ≤60 years: OR=1.118, 95% CI=0.795-1.571, p-0.522). CCL18 overexpression was associated with poor overall prognosis of breast cancer (Hazard Ratio/HR=2.969, 95% CI=1.361- 6.478, p<0.01, random effects). CONCLUSIONS: CCL18 is highly expressed in cancer tissues and is closely related to tumor metastasis and prognosis, and its role in tumor development is worth of further study.

Construction and identification of the recombinant lentiviral expression vector targeting human BAX inhibitor-1 gene.

PURPOSE: The aim of this study was to construct a recombinant lentiviral expression vector targeting human BAX inhibitor- 1(BI-1) gene and observe its expression in NIH3T3 cells. METHODS: Human BI-1 gene was amplified by polymerase chain reaction (PCR), and then cloned into the vector pLCMV- IG using DNA recombinant technique. After the inserted sequences in the recombinant plasmids were identified by PCR, and double digesting and DNA sequencing analysis, the recombinant lentivirus was packaged and administered into NIH3T3 cells. The BI-1 mRNA and protein expression were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: PCR double digesting analysis and DNA sequencing confirmed that the BI-1 DNA sequences were successfully inserted into the lentiviral vectors. After transfection with the recombinant lentivirus, BI-1 expression in NIH3T3 cells was significantly increased at both mRNA and protein levels. CONCLUSION: The lentiviral vector expressing BI-1 has been successfully constructed, which allowed for the subsequent analysis of the role of BI-1 in cell growth and transduction.

Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1alpha protein accumulation and vascular endothelial growth factor expression in human breast cancer cells.

AIM: To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7). METHODS: Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1alpha and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1alpha and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed. RESULTS: Dau significantly inhibited IGF-I-induced HIF-1alpha protein expression but had no effect on HIF-1alpha mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1alpha and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1alpha protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs. CONCLUSION: Dau inhibits human breast cancer angiogenesis by suppressing HIF-1alpha protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.

The serum level of CC chemokine ligand 18 correlates with the prognosis of non-small cell lung cancer.

BACKGROUND: CC chemokine ligand 18 (CCL18) is a chemotactic cytokine involved in the pathogenesis and progression of various cancers. Our previous research showed that the expression of CCL18 is obviously higher in non-small cell lung cancer (NSCLC) than in the adjacent normal tissues, suggesting its role in NSCLC. METHODS: We further examined the serum level of CCL18 in 80 NSCLC patients with enzyme-linked immunosorbent assay and simultaneously analyzed the survival curve of these patients by the Kaplan-Meier method, and then utilized a log-rank test to evaluate the correlation of CCL18 expression with the malignant progression of NSCLC. RESULTS: Our results showed that the median serum concentration of CCL18 was significantly elevated to 436.11 ng/mL in NSCLC patients compared to 41.97 ng/ml in healthy people (P<0.01), which was also positively related to the expression of lung cancer biomarkers carcinoma-embryonic antigen and cytokeratin fragment antigen 21-1. Moreover, correlation analysis showed that an increased level of serum CCL18 was associated with a worse survival time in NSCLC patients. CONCLUSION: Our findings suggest that the serum CCL18 level of NSCLC patients was negatively correlated with the prognosis, thus suggesting that CCL18 may serve as a potential circulating biomarker for NSCLC diagnosis.

[Effects of green tea extract on expression of human papillomavirus type 16 oncoproteins-induced hypoxia-inducible factor-1alpha and vascular endothelial growth factor in human cervical carcinoma cells].

OBJECTIVE: To investigate the effects of green tea extract (GTE) and its major component (-)-epigallocatechin-3-gallate (EGCG) on the human papillomavirus (HPV) type 16 oncoproteins (E6 and E7)-induced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human cervical carcinoma cells. METHODS: Human cervical carcinoma cells of the line C-33A were divided to 4 groups to be transiently transfected with the plasmid HA-pSG5-HPV-16E6 containing the HPV type 16 oncoprotein E6, HA-pSG5-HPV-16 E7, or empty plasmid HA-pSG5, or just exposed to the transfection reagent Lipofectamine 2000 as mock transfection control group. Western blotting and ELISA were used to detect the protein expression of HIF-1alpha and the protein expression of VEGF 18, 24, and 48 h after the transfection. Twenty-four hours after the transfection, the transfected-cells were treated with GTE (40 microg/ml) or EGCG (50 micromol/L) for 8, 16, and 24 h respectively, or treated with GTE of the concentrations of 20, 40, and 80 microg/ml or EGCG of the concentrations of 25, 50, and 100 micromol/L respectively for 16 h. The HIF-1alpha and VEGF protein levels were analyzed by Western blotting and ELISA respectively and the HIF-1alpha and VEGF mRNA levels were determined with real-time PCR. RESULTS: Twenty-four hours after transfection, the HIF-1alpha protein expression of the 16E6 and 16E7 groups were significantly decreased, and the VEGF protein levels of the 16E6 and 16E7 groups were (870+/-66) and (1487+/-51) pg/ml respectively, both significantly higher than that of the empty plasmid group [(366+/-65) pg/ml, both P<0.01]. Treated by 40 microg/ml of GTE or 50 micromol/L of EGCG for 16 h, (1) the HIF-1alpha protein levels of the 16E6 and 16E7 groups were significantly decreased; (2) the VEGF protein levels of the 16E6 group were (476+/-34) and (477+/-65) pg/ml respectively, and the VEGF mRNA levels of the 16E6 group were 1.208+/-0.196 and 1.174+/-0.208 respectively, all significantly lower than those of the untreated group [(870+/-66) pg/ml and 1.805+/-0.081 respectively, all P<0.01]; and (3) the VEGF protein levels of the 16E7 group were (649+/-55) and (514+/-37) pg/ml respectively, and the VEGF mRNA levels of the 16E7 group were 1.442+/-0.136 and 1.399+/-0.064 respectively, all significantly lower than those of untreated group [(1487+/-51) pg/ml and 2.123+/-0.120 respectively, all P<0.01]. The 80 microg/ml of GTE or 100 micromol/L of EGCG showed much stronger effects on the inhibition of VEGF protein expression than 40 microg/ml of GTE or 50 micromol/L of EGCG (both P<0.01). CONCLUSION: GTE and EGCG can remarkably inhibit the HPV-16 oncoproteins-induced expression of HIF-1alpha protein, and VEGF protein and mRNA in human cervical carcinoma cells. Moreover, GTE and EGCG decrease the VEGF protein expression dose-dependently.

[Study on the effect and its mechanisms of genistein on the cell cycle of highly metastatic ovarian carcinoma HO-8910PM cells].

OBJECTIVE: To study the effect and its possible mechanism of genistein on the cell cycle of human highly metastatic ovarian carcinoma HO-8910PM cells. METHODS: Trypan blue stain assay was used to examine the effect of genistein on proliferation of HO-8910PM cells after 24 hours treatment. The cell cycle was assessed by flowcytometry (FCM). The expression level of NF-kappaB (p65) and the level of VEGF were assessed by Western blot analysis. RESULTS: Genistein could inhibit the proliferation of HO-8910PM cell and block the cell cycle at G1 phase. The expession level of NF-kappaB (P65) protein decreased obviously in HO-8910PM cells treated with 25 approximately 100 micromol/L genistein for 24 hours, and the effect appeared in the experssion of VEGF. CONCLUSION: The effect on cell cycle of genistein is involved in the decreasing expression of NF kappaB (p65) and the level of VEGF.

[Association of mitochondrial DNA variation with type 2 diabetes mellitus].

OBJECTIVE: To explore the prevalence of mitochondrial DNA (mtDNA) mutations in patients with type 2 diabetes mellitus in Hubei. METHODS: A total of 184 cases of type 2 diabetes mellitus and 210 matched healthy controls with normal glucose tolerance were recruited for the study. The variants of mtDNA, including MIND13316 (G-->A), MIND13394 (T-->C), MTTE14693 (A-->G), MTTL1 3243 (A-->G), MTRNA1310 (C-->T) and 16189 (T-->C), were screened using PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing. The mutations were analyzed by mfold or tRNAscan-SE softwares. RESULTS: The mutation rates of 3316 (G-->A), 3394 (T-->C), 14693 (A-->G) were 3.26%, 2.72% and 2.17% respectively in type 2 diabetes group, whereas in the control group, the point mutations of 3394 (T-->C) and 14693 (A-->G) were not detected, but two subjects with 3316 (G-->A) were found (0.99%). There were significant differences in mutation rates of 3394 (T-->C) and 14693 (A-->G) between the two groups (P<0.05). In 4 of 184 cases, a T to C transition at nucleotide position 14693 was uncovered for the first time. The prevalence of 16189 variant among type 2 diabetes was significantly higher that of the controls (36.9% vs 26.6%, P=0.03). Moreover, the type 2 diabetes with 16189 variant showed higher fasting serum insulin level and higher HOMA-IR level than those without 16189 variant; stepwise multiple regression analysis showed the 16189 variant was an independent factor contributing to HOMA-IR (R(2)=0.043, P=0.037). Secondary structure prediction revealed that there were differences in 3394 T-->C vs wild-type ND1 protein and in 14693 A-->G vs wild-type tRNA(Glu) protein. CONCLUSION: The mutations of 3394 (T-->C) and 14693 (A-->G) may contribute to the genetic predisposition to type 2 diabetes; 16189 (T-->C) variant is associated with insulin resistance and risk factor of diabetes.

[Effect of proanthocyanidins on COX-2 enzyme activity and COX-2 mRNA /protein expression in LPS-induced RAW264.7 cells].

AIM: To study the effect of proanthocyanidins on the COX-2 enzyme activity and COX-2 protein expression in LPS-induced RAW264.7 cells. METHODS: After being pretreated with different concentrations of proanthocyanidins for 30 min, and then 1 mg x L(-1) LPS for 9 h, the effect of proanthocyanidins on the activity of COX-2 enzyme in RAW264.7 cells was analysed by radioimmunoassay (RIA). After being pretreated with different concentrations of proanthocyanidins for 30 min, and then 1 mg x L(-1) LPS for 9 h, the effects of proanthocyanidins on the expressions of COX-2 mRNA and protein in RAW264.7 cells were analysed by RT-PCR and Western blotting. RESULTS: The activity of COX-2 enzyme was not inhibited by proanthocyanidins (0. 8, 4 and 20 mg x L(-1), P > 0.05 vs LPS group), but the activity of COX-2 enzyme was significantly inhibited by 10 micromol x L(-01) NS-398 (P < 0.01 vs LPS group). The expression of COX-2 mRNA was inhibited by proanthocyanidins (0. 8, 4 and 20 mg x L(-1)). The expression of COX-2 protein was inhibited by proanthocyanidins (4 and 20 mg x L(-1)). CONCLUSION: Proanthocyanidins had no effect on the activity of COX-2 enzyme in LPS-induced RAW264. 7 cells. Proanthocyanidins inhibited significantly the expression of COX-2 mRNA and protein in LPS-induced RAW264.7 cells.

[Inhibitory effects of bcl-xl antisense oligodeoxynucleotides on growth of human nasopharyngeal carcinoma in nude mice].

OBJECTIVE: To investigate the inhibitory effects of bcl-xl antisense oligodeoxynucleotides (ASODN) on growth and gene expression of human nasopharyngeal carcinoma (NPC) in nude mice. METHODS: CNE-2Z cell line was implanted subcutaneously into nude mice. Twenty four h after implantation, bcl-xl ASODN and mismatch control oligonucleotides (SCODN) were injected subcutaneously into nude mice, respectively. The tumor volume and weight were measured twice weekly. The histopathological changes of the tumors were observed by HE staining. RT-PCR and Western-blot were performed for bcl-xl gene expression. RESULTS: Growth of NPC was significantly inhibited in the ASODN therapy group as compared with that in the control group (P < 0.01). The growth inhibitory rate was 41.7% in the ASODN group and 19.0% in the SCODN group. The expression level of bcl-xl mRNA and protein was decreased in the ASODN group, whereas in the SCODN group there was no significant difference in contrast with saline-treated control group. CONCLUSION: Our findings suggest that bcl-xl antisense oligodeoxynucleotides results in marked inhibition of NPC growth in nude mice. It may be a novel treatment approach for human nasopharyngeal carcinoma.

Berberine hydrochloride impact on physiological processes and modulation of twist levels in nasopharyngeal carcinoma CNE-1 cells.

OBJECTIVE: The main purpose of this work was to investigate the effect of berberine hydrochloride (BH) on the proliferation, apoptosis, migration, and invasion of CNE-1 nasopharyngeal carcinoma cells. Our results shed light on the functional components of traditional Chinese herbs for potential use in modern medicine. METHODS: The CNE-1 cell line was treated with different concentrations of BH and effects on cell viability and proliferation were evaluated using the Cell Counting Kit-8 (CCK-8) assay. Anti-migratory and anti-invasive actions of BH were investigated using wound healing assays and the Millicell Hanging cell culture insert system, respectively. Expression of the epithelial-mesenchymal transition (EMT)-related gene twist (Twist) was analyzed by real-time PCR and Western blotting. Apoptosis was estimated with an annexin-V fluorescein (FITC) apoptosis detection kit, as well as with reference to levels of activated caspase-3 of CNE-1 cells before and after treatment with BH utilizing fluorescence spectroscopy. RESULTS: BH was capable of reducing proliferation and viability of CNE-1 cells in a dose- and time-dependent manner, also demonstrating anti-migratory and anti-invasive capacities which correlated with reduction in expression of Twist. Finally, BH was able to induce significant amounts of apoptosis in CNE-1 cells, as demonstrated by an increase in the activity of caspase-3 and in annexin-V staining following treatment. CONCLUSION: BH extracted from rhizoma coptidis demonstrated an ability to block proliferation, induce apoptosis, and impair the migration and invasion of the CNE-1 cell line Considering these properties, our results suggest that BH could be an important compound for consideration in the treatment of nasopharyngeal carcinoma.

[Identification of a novel large deletion of factor subunit A mRNA associated with hereditary factor deficiency].

OBJECTIVE: To analyze the expressed mRNA of the factor subunit A (FA) in monocyte in a hereditary factor (F) deficiency family. METHODS: The F A mRNA of the proband and the other family members was analyzed by RT-PCR, semi-quantitative RT-PCR, cloning and sequencing. The three dimensional structure of the protein was predicted by SWISS-MODEL and viewed by RASMIOL. RESULTS: (1) A large in frame deletion from codons 11 to 279, spanning from exon 2 to 7 of F A (DelCD11-279), was identified in the proband at mRNA level and a truncated protein is predicted composed of 464 amino acids. Compared with the normal and the other families, the proband showed lower level of F A mRNA in RT-PCR. (2) SWISS-MODEL analysis showed that the truncated protein lacked the ß-sandwich and a part of catalytic core, resulting in loss of the normal catalytic domains. CONCLUSION: DelCD11-279 of F A mRNA is associated with hereditary F deficiency. The reduced expressing level of F A gene is one of the causes resulting in F deficiency in the patients.

Lentivirus-mediated RNA interference targeting Bax inhibitor-1 suppresses ex vivo cell proliferation and in vivo tumor growth of nasopharyngeal carcinoma.

Bax inhibitor-1 (Bi-1), an anti-apoptotic protein that belongs to the Bcl-2 family, plays an important role in the mitochondrial apoptosis pathway to suppress Bax-induced apoptosis. In several human cancers, including nasopharyngeal carcinoma, its expression was found to be increased; however, up-regulated expression of this protein has been linked to increased cell proliferations. In this study, we down-regulated the gene expression of Bi-1 in nasopharyngeal carcinoma cells by using a lentivirus transfection system packed with short hairpin RNA targeting Bi-1 and used an in vivo model to assess its efficacy as a target in human gene therapy. The data indicated that human malignant nasopharyngeal carcinoma cells, CNE-1 and SUNE-1, transfected with lentiviral short hairpin RNA targeting Bi-1 grew more slowly and showed a higher degree of apoptosis. Moreover, the tumorigenicity of CNE-1 was significantly suppressed when inoculated mice were intratumorically injected with the same vector. Taken together, these data lead us to conclude that Bi-1 plays a crucial role in CNE-1 tumorigenesis and that Bi-1 may be a novel therapeutic target for nasopharyngeal carcinoma.

[Correlation of PI3K-Akt signal pathway to apoptosis of tumor cells].

PI3K-Akt signal pathway is an important intracellular signal transduction pathway. It plays important roles in cell apoptosis and survival by affecting the activity of downstream effector molecules, and it is closely associated with the development and progression of human tumors, therefore, PI3K and Akt might be potential targets for tumor therapy. This article reviewed recent progress on the composition, function, regulation and anti-apoptosis of PI3K-Akt signal pathway, and discussed its potential application to tumor gene therapy.

[Determination of the trace levels of urinary fibrinopeptides by high-performance capillary electrophoresis].

OBJECTIVE: To establish a high-performance capillary electrophoresis (HPCE)-based method for detection of trace amount of urinary fibrinopeptide A and B (FPA and FPB, respectively) as the specific molecular markers of thrombus formation in vivo. METHODS: The HPCE system consisted of a 25 cm x 50 microm (inner diameter) coated capillary column, 0.1 mol/L phosphoric acid buffer (pH 2.5) and a UV-detector (wavelength at 190 nm). To improve the sensitivity and reproducibility, solid-phase extraction of FPA and FPB in the urine was performed using a Sep-pak C18 column, with a synthetical fibrinopeptide B-Tyr (FPB-Tyr) as the internal standard. RESULTS: With this HPCE method, optimal separations of FPA, FPB and FPB-Tyr was achieved within 16 min, with the migration time of 7.28 min, 14.31 min and 15.22 min, respectively. The adjusted peak area ratios of FPA or FPB and the internal standard showed good linearity with the corresponding concentrations of FPA or FPB spiked in the urine(R>0.99). Under the above chromatography conditions, the minimum detection concentration of FPA and FPB in untreated urine was 30 microg/L and 40 microg/L, respectively, and the assay precision and recovery of FPA and FPB were acceptable. CONCLUSION: The method we established is reliable and specific for separation and identification of fibrinopeptides and other bioactive peptides.

[Inhibitory effects of tumor suppressor gene PTEN on proliferation and metastasis of breast cancer ZR-75-1 cells].

BACKGROUND & OBJECTIVE: Tumor suppressor gene PTEN could not only inhibit the proliferation of cancer cells, but also inhibit their metastasis. However, the mechanism is still unclear. This study was to investigate the effects of PTEN gene on the proliferation and metastasis of human breast cancer ZR-75-1 cells, and explore the mechanisms. METHODS: Wild-type PTEN (wt-PTEN) plasmid and phosphatase-defective PTEN (G129R-PTEN) plasmid were transfected into ZR-75-1 cells by liposome, respectively. Cell proliferation was detected by MTT assay. Transfected cells were selected by puromycin. The expression of PTEN protein was detected by Western blot. Cell adhesion and invasion were tested by adhesion test and invasion test. RESULTS: The proliferation inhibition rate was significantly higher in wt-PTEN-transfected ZR-75-1 cells than in untransfected cells and G129R-PTEN-transfected cells (42.7% vs. 0% and 2.7%, P<0.01); there was no significant difference between untransfected cells and G129R-PTEN-transfected cells(P>0.05). The proliferation inhibition of ZR-75-1 cells was enhanced along with the increase of culture time and concentration of wt-PTEN. wt-PTEN also induced cell apoptosis. PTEN protein was expressed efficiently in the cells transfected with either wt-PTEN or G129R-PTEN. The inhibition rates of adhesion and invasion were significantly higher in wt-PTEN-transfected cells than in G129R-PTEN-transfected cells (65.7% vs. 8.8%, 70.4% vs. 6.9%, P<0.01). CONCLUSION: Wild-type PTEN gene with dual-specific phosphatase activity can inhibit the proliferation and metastasis of ZR-75-1 cells.

RNA interference by expression of short hairpin RNAs suppresses bcl-xL gene expression in nasopharyngeal carcinoma cells.

AIM: To evaluate a new plasmid mediated RNA interference (RNAi) system and investigate whether knock-down of bcl-xL by short hairpin RNA (shRNA) can induce apoptosis of human nasopharyngeal carcinoma (NPC) cell line CNE-2Z in vitro. METHODS: The plasmid containing mU6 promoter was subcloned to yield the pmU6 plasmid, recombinant plasmid expressing shRNA targeting bcl-xL gene was designed and constructed, and were co-transfected cells with green fluorescence protein expressing plasmid. Flow cytometry was used to evaluate transfection efficiency, and RT-PCR and Western blot were applied to analyze bcl-xL mRNA and protein levels, respectively. RESULTS: The shRNA expressed by the recombinant plasmid efficiently suppressed bcl-xL gene expression and induced apoptosis of NPC cells in vitro. CONCLUSION: The recombinant plasmid can sufficiently mediate RNAi in CNE-2Z cells, and knock-down of the bcl-xL expression by shRNA significantly induced apoptosis in CNE-2Z cells. The results suggest this new system, mediated RNAi can be used as a tool for the study of gene function and gene therapy.

Knockdown of survivin expression by small interfering RNA induces apoptosis in human breast carcinoma cell line MCF-7.

BACKGROUND & OBJECTIVE: survivin, a member of inhibitor of apoptosis protein (IAP) gene family, expresses in various human cancer tissues, and may facilitate tumor cell evasion from apoptosis, and promote aberrant mitotic progression. This study was to investigate cell proliferation and apoptosis status of human breast carcinoma cell line MCF-7 after knockdown of survivin. METHODS: Small interfering RNA was transfected into MCF-7 cells to inhibit expression of survivin. mRNA and protein levels of survivin were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Proliferation inhibition rate of MCF-7 cells was analyzed by MTT assay. Cell apoptosis was assayed by flow cytometry (FCM). RESULTS: The expression of survivin in siRNA-transfected group decreased by 64% in comparison to untransfected group. After treatment of different concentrations of siRNA, proliferation inhibition rate and apoptosis rate of MCF-7 cells were increased. The highest proliferation inhibition rate was 60.9%, and the highest apoptosis rate was 29.0% after treatment of 200 nmol/L of siRNA. CONCLUSION: survivin siRNA might be a useful therapeutic agent for the treatment of breast carcinoma.

Inducing apoptosis of human nasopharyngeal carcinoma cell line CNE-2Z by bcl-xL short hairpin RNA.

BACKGROUND & OBJECTIVE: Recent studies showed overexpression of bcl-x(L) in human nasopharyngeal carcinoma (NPC) cell line CNE-2Z; it may play a pivotal role in tumorigenesis, metastasis, and drug resistance of NPC. This study was to explore inducing effect of bcl-x(L) short hairpin RNA (shRNA) on apoptosis of CNE-2Z cells. METHODS: After transfection of recombinant plasmid pmU6-RNAi expressing bcl-x(L) shRNA, apoptotic CNE-2Z cells were detected by fluorescent staining and flow cytometry (FCM). mRNA levels of bcl-x(L), bcl-2, survivin, and caspase-3 was detected by reverse transcription-polymerase chain reaction (RT-PCR); while protein levels of Bcl-x(L), Caspase-3, and P53 were detected by Western blot. RESULTS: When treated with pmU6-RNAi for 24 h, an obvious apoptotic peak of CNE-2Z cells appeared; cell shrinkage, chromatin condensation, and nuclear fragmentation were observed in most cells under fluorescent microscope. RT-PCR analysis showed that pmU6-RNAi down-regulated mRNA levels of bcl-x(L), bcl-2, and caspase-3, but had little or no effect on mRNA level of survivin; Western blot analysis showed an obvious reduction in protein levels of Bcl-x(L) and Caspase-3, and a great increase in protein level of P53. CONCLUSIONS: bcl-x(L) shRNA can induce apoptosis of CNE-2Z cells, which may be closely related to down-regulation of bcl-2, caspase-3 and p53. bcl-x(L) shRNA may be helpful for developing gene therapy for NPC.

[Knockdown of bcl-xL expression with RNA interference induces nasopharyngeal carcinoma cells apoptosis].

OBJECTIVE: To study the inhibition of the expression of bcl-xL gene induced by RNA interference in CNE-2Z cell line in addition to the inhibition of its proliferation and apoptotic induction. METHODS: Small interfering RNAs targeting bcl-xL gene were synthesized by using web design software provided by Amnion and the silencer short interfering RNA (siRNA) construction kit; fluorescein-labeled siRNAs were done by FAM-silencer siRNA labeling kit; siRNAs were transfected into CNE-2Z cells by using lipofectamine 2000 reagent; siRNA transfection efficiencies were analyzed by fluorescent microscopy; down-regulation of bcl-xL was detected by RT-PCR; thiazolyl blue (MTT) assay was used to assess the cell growth; apoptosis of CNE-2Z cells was analyzed by flow cytometry. RESULTS: Green fluorescence in the cells was seen clearly in FAM-labeled siRNA transfected group under the fluorescent microscope while none in the untransfected group. Different down-regulations of bcl-xL mRNA expression were found in the transfected groups. The expression of bcl-xL mRNA decreased by 10% - 70% in the siRNAs transfected CNE-2Z by RT-PCR scan analysis. The inhibitory rate of cell proliferation depended on time and concentrations to some extent. Different cell apoptosis could be induced by different concentrations of siRNA4. CONCLUSIONS: The synthesized siRNAs in vitro were able to down-regulate the expression of bcl-xL There were different capabilities of the specific siRNAs down-regulation. The transient transfected bcl-xL siRNA4 could effectively inhibit the growth of the cancer cells and induce theirs apoptosis. It was suggested that the siRNA technique provide not only an extremely powerful tool for the functional analysis of genome but also a new method for anti-nasopharyngeal carcinoma gene therapy.

[Induction of apoptosis in human nasopharyngeal carcinoma cell line CNE-2Z by curcumin].

BACKGROUND & OBJECTIVE: Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China. Radiotherapy is a major therapy for NPC in early phase,while proper chemotherapy is necessary for NPC in late phase. Some chemotherapeutic drugs have been used to treat tumors by inducing apoptosis of tumor cells. This study was to investigate effects of curcumin on proliferation,and apoptosis of NPC cell line CNE-2Z. METHODS: CNE-2Z cells were treated with different concentrations of curcumin, inhibition rates of cell proliferation,and IC(50) of curcumin were detected by MTT method. Cell apoptosis was analyzed by flow cytometry (FCM), agarose gel electrophoresis,and Hoechst33258-PI fluorescence staining. RESULTS: Proliferation of CNE-2Z cells was inhibited obviously by curcumin in dose- and time-dependent manners. IC(50) of curcumin to CNE-2Z cells at 24,48,and 72 h were (24.05+/-0.47),(19.20+/-0.17),and (7.35+/-0.50) micromol/L, respectively. After treated with 5,10,and 20 micromol/L of curcumin for 24 h, apoptotic rates of CNE-2Z cells were (4.9+/-3.2)%,(10.7+/-2.7)%, and (14.7+/-0.5)%, respectively. After treated with 10,20 micromol/L of curcumin for 24 h, morphologic changes, such as chromatin shrinkage,nuclear condensation,and chromatin fragmentation,were observed in CNE-2Z cells by fluorescent staining, a fragmented DNA ladder was detected by electrophoresis. CONCLUSION: Curcumin may induce apoptosis, and inhibit proliferation of CNE-2Z cells.