OBJECTIVE: To investigate the effects of mTOR inhibitors everolimus (EVE) and gemcitabine (GEM) on the proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma (DLBCL) cell line U2932, and further explore the molecular mechanisms, so as to provide new ideas and experimental basis for the clinical treatment of DLBCL. METHODS: The effect of EVE and GEM on the proliferation of U2932 cells was detected by CCK-8 assay, the IC₅₀ of the two drugs was calculated, and the combination index (CI=) of the two drugs was calculated by CompuSyn software. The effect of EVE and GEM on apoptosis of U2932 cells was detected by flow cytometry with AnnexinV-FITC/PI staining. Flow cytometry with propidium iodide (PI) staining was used to detect the effect of EVE and GEM on the cell cycle of U2932 cells. Western blot assay was used to detect the effects of EVE and GEM on the channel proteins p-mTOR and p-4EBP1, the anti-apoptotic proteins MCL-1 and Survivin, and the cell cycle protein Cyclin D1. RESULTS: Both EVE and GEM could significantly inhitbit the proliferation of U2932 cells in a time- and dose-dependent manner (r=0.465, 0.848; 0.555, 0.796). According to the calculation of CompuSyn software, EVE combined with GEM inhibited the proliferation of U2932 cells at 24, 48 and 72 h with CI=<1, which had a synergistic effect. After treated U2932 cells with 10 nmol/L EVE, 250 nmol/L GEM alone and in combination for 48 h, both EVE and GEM induced apoptosis, and the difference was statistically significant compared with the control group (P<0.05). The apoptosis rate was significantly enhanced after EVE in combination with GEM compared with single-agent (P<0.05). Both EVE and GEM alone and in combination significantly increased the proportion of cells in G₁ phase compared with the control group (P<0.05). The proportion of cells in G₁ phase was significantly increased when the two drugs were combined (P<0.05). The expression of p-mTOR and effector protein p-4EBP1 was significantly downregulated in the EVE combined with GEM group, the expression of anti-apoptotic proteins MCL-1, Survivin and cell cycle protein cyclin D1 was downregulated too (P<0.05). CONCLUSION EVE combined with GEM can synergistically inhibit the proliferation of U2932 cells, and the mechanism may be that they can synergistically induce apoptosis by downregulating the expression of MCL-1 and Survivin proteins and block the cell cycle progression by downregulating the expression of Cyclin D1.
Humans ; Gemcitabine ; Everolimus/pharmacology* ; Survivin/pharmacology* ; Cyclin D1/pharmacology* ; Myeloid Cell Leukemia Sequence 1 Protein ; Cell Line, Tumor ; Cell Proliferation ; TOR Serine-Threonine Kinases ; Apoptosis ; Apoptosis Regulatory Proteins ; Cell Cycle Proteins ; Lymphoma, Large B-Cell, Diffuse

Rats ; Animals ; Urinary Bladder Neck Obstruction/pathology* ; Survivin/genetics* ; Urinary Bladder/pathology* ; Fibrosis

The aim of this study was to investigate the effect of Wnt5a on the vincristine (VCR) resistance in human ovarian carcinoma SKOV3 cells and its possible mechanism. The drug-resistant SKOV3/VCR cells were established by stepwise exposure to VCR, and then the SKOV3/VCR cells were stably transfected with specific shRNA interference plasmid vector targeting for Wnt5a. The mRNA expression level of Wnt5a was measured by RT-PCR. CCK-8 assay was used to detect the cell viability of SKOV3/VCR cells. The apoptosis was analyzed by flow cytometry. The protein expression levels of Wnt5a, MDR1, Survivin, β-catenin, Akt, p-Akt(S473), GSK3β and p-GSK3β(Ser9) were detected by Western blot. The result showed that SKOV3/VCR cells had significantly higher protein expression levels of Wnt5a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5a, compared with SKOV3 cells (P < 0.05). WNT5A gene silencing significantly increased the sensitivity of SKOV3/VCR cells to VCR, the IC of VCR being decreased from 38.412 to 9.283 mg/L (P < 0.05), synergistically enhanced VCR-induced apoptosis of SKOV3/VCR cells (P < 0.05), down-regulated the protein expression levels of MDR1, β-catenin and Survivin (P < 0.05), and inhibited phosphorylation of Akt and GSK3β (P < 0.05). Meanwhile, LY294002 (PI3K inhibitor) decreased the protein expression levels of MDR1, β-catenin and Survivin, as well as the phosphorylation levels of Akt and GSK3β in SKOV3/VCR cells (P < 0.05). These results suggest that WNT5A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.
ATP Binding Cassette Transporter, Subfamily B ; metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Female ; Gene Silencing ; Glycogen Synthase Kinase 3 beta ; metabolism ; Humans ; Ovarian Neoplasms ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Survivin ; metabolism ; Vincristine ; pharmacology ; Wnt-5a Protein ; metabolism

OBJECTIVE: To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms. METHODS: MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot. RESULTS: The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group. CONCLUSION MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.
Apoptosis ; Bone Marrow Cells ; Caspase 3 ; Cell Proliferation ; Daunorubicin ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; Mesenchymal Stem Cells ; Survivin

OBJECTIVE: To study the effect of knocking down fascin on cervical cancer cell proliferation and tumorigenicity in nude mice. METHODS: Cervical cancer CaSki cells were infected with a lentiviral vector carrying fascin siRNA or with a negative control lentivirus, and fascin mRNA and protein expressions in the cells were detected using qRT-PCR and Western blotting. MTT assay was used to determine the proliferation of CaSki cells with fascin knockdown. CaSki cells transfected with fascin siRNA or the control lentiviral vector and non-transfected CaSki cells were inoculated subcutaneously in nude mice, and the volume and weight of the transplanted tumor were measured; Western blotting was used to detect the expressions of proliferating cell nuclear antigen (PCNA), survivin, cyclin dependent kinase 4 (CDK4) and p21 proteins in the tumor xenograft. RESULTS: Infection with the lentiviral vector carrying fascin siRNA, but not the negative control vector, caused significant reductions in the expression levels of fascin mRNA and protein in CaSki cells ( < 0.05). Fascin knockdown resulted in significantly reduced proliferation of CaSki cells ( < 0.05). The nude mice inoculated with CaSki cells with fascin knockdown showed reduced tumor volume and weight, lowered levels of PCNA, survivin and CDK4, and increased expression of p21 protein in the tumor xenograft compared with the control mice. The negative control lentivirus did not affect the proliferation or tumorigenicity of CaSki cells in nude mice or the expression levels of PCNA, survivin, CDK4 or p21 proteins in the xenografts. CONCLUSIONS Knocking down fascin can inhibit the growth and tumorigenicity of cervical cancer cells in nude mice.
Animals ; Apoptosis ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Female ; Gene Knockdown Techniques ; Genetic Vectors ; Humans ; Mice ; Mice, Nude ; Microfilament Proteins ; genetics ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Survivin ; metabolism ; Transfection ; Tumor Burden ; Uterine Cervical Neoplasms ; etiology ; pathology

OBJECTIVE: To explore the relationship of the local recurrence with the expression of protein Survivin and MMP-2 in the primary lesions and the surgical margins of laryngeal carcinoma. METHOD: The primary lesions and the surgical margins of laryngeal carcinoma of 48 patients were made into serial sections. Immunochemical methods was used to detect the expression of protein Survivin and MMP-2 in the primary lesion and the surgical margins of laryngeal carcinoma of 48 patients. RESULT: The positive expression for Survivin and MMP-2 in the primary lesion was 70.83% (34/48) and 66.67% (32/48) respectively, and the positive expression of Survivin and MMP-2 in the surgical margins of laryngeal carcinoma was 47.92% (23/48) and 37.50% (18/48), which in the primary lesion was significantly higher than those of the surgical margins of laryngeal carcinoma (P < 0.05). The recurrence rates of primary lesion positive for Survivin (34 cases) and MMP-2 (32 cases) were 26.47% (9/34) and 25.00% (8/32), which were higher than negative for them 7.14%(1/14) and 12.50% (2/16) (P > 0.05). The recurrence rates of those with Survivin (23 cases) and MMP-2 (18 cases) positive surgical margins were 34.78% (8/23) and 38.89% (7/18) respectively, which were significantly higher than those with negative ones 8.00% (2/25) and 10.00% (3/30) (P < 0.05). Logistic analysis showed that the expression of Survivin and MMP-2 protein in the surgical margins of laryngeal carcinoma was positively associated with the recurrence rates. CONCLUSION Laryngeal carcinoma patients with Survivin-positive or MMP-2-positive margin would have a higher recurrence rate. Survivin and MMP-2 protein can be used as biomarkers for local recurrence of laryngeal carcinoma after operation.
Biomarkers, Tumor ; metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Laryngeal Neoplasms ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Recurrence, Local ; Survivin

OBJECTIVE: To investigate the expression of Survivin, p53 and Ki67 in laryngeal carcinoma and the relation with clinical data. METHOD: Immunohistochemical staining (SP) was used to detect expression of Survivin, p53 and Ki67 of 64 cases with laryngeal carcinoma, 26 cases with precancerosis, 34 cases with vocal polyps. RESULT: The positive expression rates of Survivin, p53 and Ki67 were 59.4%, 68.8%, 65.6% respectively in laryngeal carcinoma, which were significantly higher than those in precancerosis and vocal polyps (P < 0.01). The expression of Survivin, p53 and Ki67 in laryngeal carcinoma were significantly statistical different in TNM stage and lymph node metastasis (P < 0.05), but were not correlated with patients' ages, the pathological grades, 3 years and 5 years surviving rates (P > 0.05). The expression of Survivin, Ki-67 and p53 was positively correlated (r = 0.607, 0.541, 0.648, P < 0.01) in laryngeal carcinoma. CONCLUSION Survivin, p53 and Ki-67 may play an important role in the carcinogenesis and progress of laryngeal carcinoma. They may play synergetic roles in the process of carcinogenesis of laryngeal carcinoma.
Carcinoma, Squamous Cell ; metabolism ; Humans ; Immunohistochemistry ; Inhibitor of Apoptosis Proteins ; metabolism ; Ki-67 Antigen ; metabolism ; Laryngeal Neoplasms ; metabolism ; Larynx ; Lymphatic Metastasis ; Polyps ; metabolism ; Survivin ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVE: This study aimed to explore the relationship among expression of Survivin and MRP and drug resistance in NPC. METHOD: Expression of Survivin were detected by immunohistochemistry method in 45 cases of NPC and 24 cases of normal mucous membrane of nasopharynx (NMMN). The relationship between expression of Survivin and pathological factors in NPC were analysized. Expression of Survivin and MRP were detected in 31 patients of NPC with paclitaxel resistance and 20 patients of NPC without paclitaxel resistance. The relation- ship among the expression of Survivin or MRP and paclitaxel resistance in NPC were analysized. The paclitaxel resistance cell line, 5-8F-PTX(+); was established by a step-increased method. The expression of Survivin and MRP were detected by western blot in 5-8F-PTX(+) and 5-8F. RESULT: The positive were 71. 1% (32/45) in NPC and 8.33% (2/24) in NMMN. And there were significantly differences between them (P < .05). There were relationship among expression of Survivin and differentiation degree, lymph node metastasis, distant metastasis, and clinic stages of NPC. The positive were 75.9% (31/39) in moderately differentiated NPC and 16.7% (1/6) in lowly differentiated NPC, respectively. There were significantly differences between them (P < 0.05). The positive of Survivin were 83.9% (26/31) in NPC patients with paclitaxel resistance and 45.0% (9/20) in NPC patients without Paclitaxel resistance, respectively. There were significantly differences between them (P < 0.05). The positive of MRP were 87.1% (27/31) in NPC patients with paclitaxel resistance and 40.0% (8/20) in NPC patients without paclitaxel resistance, respectively. There were significantly differences between them (P < 0.05). There were positive correlation between the expression of Survivin and MRP in NPC patients with Paclitaxel resistance. The expression of Survivin and MRP were higher in 5-8F-PTX(+) than in 5-8F. The IC50 of paclitaxel, cDDP, 5-FU and Vincristine were significantly higher in 5-8F-PTX(+) than in 5-8F. CONCLUSION There were relationship among the expression of Survivin and difference, metastasis and TNM stages of NPC. Survivin may serves as a molecular marker for development and progress in NPC. There were relationship among the high expression of Survivin and MRP and increasing of drug resistance in NPC.
Antineoplastic Agents ; pharmacology ; Carcinoma ; Cisplatin ; Drug Resistance, Neoplasm ; Fluorouracil ; Humans ; Immunohistochemistry ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Lymphatic Metastasis ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; Nasopharynx ; metabolism ; Paclitaxel ; pharmacology ; Survivin ; Vincristine

OBJECTIVE: To study the combined transfection of the siRNA target for Hif-lα and Survivin gene to human NPC CNE-2 cell and its effects on the proliferation and cycle of this cell. METHOD: Combined transfection of the siRNA target for Hif-lα and Survivin gene to human NPC CNE-2 cell, these plasmids were respectively transfected into the same cells. Cell proliferation was detected with MTT assay. The inhibitory effects on target genes were evaluated with RT-PCR and Western Blot at the levels of mRNA and protein, respectively. RESULT: MTT showed that CNE-2 cell proliferation in multi-gene plasmid group was more significantly inhibited than a single gene. The expression of mRNA and protein of two different genes were both decreased in HS group, and the interference effect of multiple genes was better than the single-gene(P<0.01). CONCLUSION HS group could restrain cell proliferation and interference the mRNA and protein expression in nasopharyngeal carcinoma CNE-2 cell, which was better than the other groups.
Apoptosis ; Carcinoma ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Inhibitor of Apoptosis Proteins ; genetics ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; Plasmids ; RNA, Messenger ; RNA, Small Interfering ; Survivin ; Transfection

OBJECTIVE: To investigate the effect of adenovirus-mediated ING4 with RGD on proliferation, apoptosis and cell cycle of human nasopharyngeal carcinoma cell CNE and explore its probable mechanism. METHOD: CNE cells were infected with Ad. RGD-ING4 and adenovirus vector, ING4 gene expression level was detected by RT-PCR and the target protein expression was tested by Western blot. MTT assay was adopted to evaluate the efect of ING4 on cell growth of CNE, Annexin -V-PE/7-AAD Double staining was used to measure the efect of ING4 on apoptosis, and PI staining was used to measure the efect of ING4 on the cell cycle. Differential expression of P21, Bcl-2 and Bax gene was detected by RT-PCR,and Differential expression of Survivin and Caspase 3 protein was detected by Western blot. RESULT: CNE cells were cultured with Ad. RGD-ING4 for 72 h ,the results showed that ING4 was overexpressed in CNE cells ,the growth of CNE cells was obviously inhibited , apoptosis rate was significantly increased and G2/M phase was arrested apparently. The results of RT-PCR showed that Ad. RGD-ING4 significantly down-regulated the Bcl-2 and up-regulates the Bax and P21 expression in CNE cells, and the difference was statistically significant(P < 0.01). Western blot showed that the expression of Survivin was decreased and Cleaved-Caspase 3 was increased. CONCLUSION Ad. RGD-ING4 can play the role of tumor suppressor synergies on nasopharyngeal carcinoma cell CNE by down-regulating Bcl-2, Survivin expression and up-regulating P21, Bax and Cleaved-Caspase 3 expression.
Adenoviridae ; Apoptosis ; Carcinoma ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Cycle Proteins ; genetics ; therapeutic use ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression ; Genetic Vectors ; Homeodomain Proteins ; genetics ; therapeutic use ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; therapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Survivin ; Tumor Suppressor Proteins ; genetics ; therapeutic use