Effect of Survivin Down-Regulation by Egr1-Survivin shRNA Combined with Radiotherapy on Radiosensitivity of Esophageal Squamous Carcinoma / Efecto de la Regulación Negativa de Survivina por shRNA de Egr1-Survivina Combinado con Radioterapia sobre la Radiosensibilidad del Carcinoma Escamoso de Esófago

SUMMARY: This study is to investigate the effect of survivin down-regulation by Egr1-survivin shRNA combined with radiotherapy on the apoptosis and radiosensitivity of esophageal squamous cell carcinoma ECA109 and KYSE150 cells. ECA109 and KYSE150 cells were transfected with Egr1-survivin shRNA, and then treated with radiotherapy. After 24 h, the mRNA and protein levels of Egr1-survivin were detected by qPCR and Western-Blot. Cell cycle and apoptosis were detected by flow cytometry. Western blot also detected levels of cleavaged Caspase 3 and Caspase 9. YM155 was used as a positive control to inhibit survivin expression. The levels of survivin mRNA and protein in ECA109 and KYSE150 cells treated with Egr1-survivin shRNA combined with radiotherapy were significantly lower than those of the blank control group, the empty vector control group, and, the YM155 + radiotherapy group (P<0.05). Meanwhile, after survivin down-regulation, the ratio of G2 to S phase of ECA109 and KYSE150 cells increased significantly, leading to significant G2 and S phase arrest. Additionally, apoptosis of ECA109 and KYSE150 cells increased significantly (P <0.01). Further, protein levels of cleavaged Caspase 3 and Caspase 9 significantly increased in Egr1-survivin shRNA combined with radiotherapy group. Egr1-survivin shRNA combined with radiotherapy can down-regulate survivin expression, which further increases the apoptosis, and enhances the radiosensitivity of ECA109 and KYSE150 cells.

Este estudio tuvo como objetivo investigar el efecto de la regulación negativa de survivina por el shRNA de Egr1-survivina combinado con radioterapia sobre la apoptosis y la radiosensibilidad del carcinoma de células escamosas de esófago Células ECA109 y KYSE150. Las células ECA109 y KYSE150 se transfectaron con shRNA de survivina Egr1 y luego se trataron con radioterapia. Después de 24 h, los niveles de ARNm y proteína de Egr1-survivina se detectaron mediante qPCR y Western-Blot. El ciclo celular y la apoptosis se detectaron mediante citometría de flujo. La transferencia Western también detectó niveles de Caspasa 3 y Caspasa 9 escindidas. Se usó YM155 como control positivo para inhibir la expresión de survivina. Los niveles de ARNm y proteína de survivina en células ECA109 y KYSE150 tratadas con shRNA de survivina Egr1 combinado con radioterapia fueron significativamente más bajos que los del grupo control en blanco, el grupo control de vector vacío y el grupo de radioterapia YM155 + (P <0,05). Mientras tanto, después de la regulación negativa de survivina, la proporción entre las fases G2 y S de las células ECA109 y KYSE150 aumentó significativamente, lo que llevó a una detención significativa de las fases G2 y S. Además, la apoptosis de las células ECA109 y KYSE150 aumentó significativamente (P <0,01). Además, los niveles de proteína de Caspasa 3 y Caspasa 9 escindidas aumentaron significativamente en el shRNA de Egr1- survivina combinado con el grupo de radioterapia. El shRNA de survivina de Egr1 combinado con radioterapia puede regular negativamente la expresión de survivina, lo que aumenta aún más la apoptosis y mejora la radiosensibilidad de las células ECA109 y KYSE150.

Synergistic Antitumor Effect of Everolimus Combined with Gemcitabine on Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

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.

The role of Survivin as a biomarker and potential prognostic factor for breast cancer

SUMMARY Breast cancer (BC) is one of the primary health problems worldwide. As the most common cancer in women in the world and in Brasil, behind only non-melanoma skin cancer, this neoplasm corresponds to approximately 28% of new cases per year in the country. BC also affects men, although the incidence corresponds to only 1% of total cases. Currently, most of the chemotherapeutic agents used in BC treatment are extremely toxic and cause long-term side effects. There is also a need to obtain earlier diagnoses, more accurate prognoses and make new therapies available that are more selective and effective in order to improve the current scenario. Therefore, this work sought to evaluate the importance of the biomarker survivin (Sur) in relation to BC, through the detailing of the role of Sur as a biomarker, the correlation between this protein and the prognosis of BC patients, and a summary of therapeutic strategies that target Sur for the development of new anticancer therapies.

RESUMO O câncer de mama (CM) é um dos principais problemas de saúde em todo o mundo. Como o câncer mais comum em mulheres no mundo e no Brasil, precedido apenas pelo câncer de pele não melanoma, essa neoplasia corresponde a aproximadamente 28% dos novos casos por ano no país. O CM também afeta homens, embora a incidência corresponda a apenas 1% do total de casos. Atualmente, a maioria dos agentes quimioterápicos utilizados no tratamento do CM são extremamente tóxicos e causam efeitos colaterais a longo prazo. Há também a necessidade de se obterem diagnósticos mais precoces, prognósticos mais precisos e disponibilizar novas terapias seletivas e efetivas, a fim de melhorar o cenário atual. Portanto, este trabalho buscou avaliar a importância do biomarcador Survivina (Sur) em relação ao CM, por meio do detalhamento do papel do Sur como biomarcador, da correlação entre essa proteína com o prognóstico de pacientes com CM e de um resumo do tratamento terapêutico e das estratégias que visam utilizar a Sur para o desenvolvimento de novas terapias anticâncer.

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin / 中国实验血液学杂志

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.

Wnt5a modulates vincristine resistance through PI3K/Akt/GSK3β signaling pathway in human ovarian carcinoma SKOV3/VCR cells / 生理学报

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.

Evaluation of survivin immunoexpression in the differentiation of high- and low-grade breast ductal carcinoma in situ / Expressão da proteína survivina em carcinoma ductal in situ de mama de alto e de baixo grau

ABSTRACT Objective To evaluate the expression of survivin protein in low- and high-grade ductal carcinoma in situ. Methods Breast tissue fragments obtained by incisional biopsy and surgical procedures of 37 women with ductal carcinoma in situ of the breast were subdivided into two groups: Group A, composed of women with low-grade ductal carcinoma in situ, and Group B, women with high-grade ductal carcinoma in situ. Survivin protein expression test was performed by immunohistochemistry, using a monoclonal antibody clone I2C4. The criterion to evaluate survivin immunoexpression was based on the percentage of neoplastic cells that presented brown-gold staining. This criterion was positive when the percentage of stained cells was ≥10%. Results The survivin protein was expressed in 22 out of 24 cases of high-grade ductal carcinoma in situ (78%), whereas, in Group A, of low-grade ductal carcinoma in situ (n=13), it was positive in only 6 cases (21.40%; p=0.004). Conclusion The frequency of expression of survivin was significantly higher in the group of patients with high-grade ductal carcinoma in situ compared to those in the low-grade ductal carcinoma in situ group.

RESUMO Objetivo Avaliar a imunoexpressão da proteína survivina nos carcinomas ductais in situ de mama de baixo e de alto graus. Métodos Fragmentos de tecido mamários obtidos por biópsia incisional e procedimentos cirúrgicos de 37 mulheres acometidas por carcinoma ductal in situ de mama foram subdivididos em dois grupos: Grupo A, formado por mulheres com carcinoma ductal in situ de baixo grau; e Grupo B, por mulheres com carcinoma ductal in situ de alto grau. A pesquisa de expressão da proteína survivina foi realizada pela técnica de imuno-histoquímica, utilizando-se anticorpo monoclonal clone I2C4. O critério de avaliação da imunoexpressão da survivina baseou-se na percentagem de células neoplásicas que apresentava coloração castanho-dourada. Considerouse tal critério positivo quando a percentagem de células apresentasse marcação ≥10%. Resultados A proteína survivina apresentou-se expressa em 22 dos 24 casos de carcinoma ductal in situ de alto grau (78%), enquanto no Grupo A, de carcinoma ductal in situ de baixo grau (n=13), apresentou-se positiva em apenas 6 casos (21,40%; p=0,004). Conclusão O índice de frequência de expressão da survivina foi significativamente mais elevado no grupo de pacientes com carcinoma ductal in situ de alto grau, quando comparado às do grupo com carcinoma ductal in situ de baixo grau.

Knocking down fascin inhibits cervical cancer cell proliferation and tumorigenesis in nude mice / 南方医科大学学报

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.

Expression of e-cadherin and wnt pathway proteins betacatenin, apc, tcf-4 and survivin in gastric adenocarcinoma: clinical and pathological implication / Expressão da e-caderina e das proteínas da via de sinalização wnt betacatenina, apc, tcf-4 e survivina no adenocarcinoma gástrico: implicações clínica e patológica

ABSTRACT Background: Gastric cancer is the fifth most frequent cancer and the third most common cause of cancer-related deaths worldwide.It has been reported that Wnt/ betacatenin pathway is activated in 30-50% of these tumors. However,the deregulation of this pathway has not been fully elucidated. Aim: To determine the expression of E-cadherin, betacatenin, APC, TCF-4 and survivin proteins in gastric adenocarcinoma tissues and correlate with clinical and pathological parameters. Method: Seventy-one patients with gastric adenocarcinoma undergoing gastrectomy were enrolled. The expression of E-cadherin, betacatenin, APC, TCF-4 and survivin proteins was detected by immunohistochemistryand related to the clinical and pathological parameters. Results: The expression rates of E-cadherin in the membrane was 3%; betacatenin in the cytoplasm and nucleus were 23,4% and 3,1% respectively; APC in the cytoplasm was 94,6%; TCF-4 in the nucleus was 19,4%; and survivin in the nucleus 93,9%. The expression rate of E-cadherin was correlated with older patients (p=0,007), while betacatenin with tumors <5 cm (p=0,041) and APC with proximal tumors (p=0,047). Moreover, the expression of TCF-4 was significantly higher in the diffuse type (p=0,017) and T4 tumors (p=0,002). Conclusion: The Wnt/betacatenin is not involved in gastric carcinogenesis. However, the high frequency of survivin allows to suggest that other signaling pathways must be involved in the transformation of gastric tissue.

RESUMO Racional: O câncer gástrico encontra-se entre as principais neoplasias malignas do mundo sendo o quinto mais incidente e o terceiro em relação ao índice de mortalidade. Acredita-se que a via Wnt/betacatenina esteja ativada em 30-50% desses tumores, porém a desregulação dela ainda não está completamente esclarecida. Objetivo: Avaliar a imunoexpressão das proteínas E-caderina, betacatenina, APC, TCF-4 e survivina em tecidos de adenocarcinoma gástrico e correlacioná-las com as variáveis clínicas dos doentes e anatomopatológicas do tumor. Método: Foram coletados os dados clínicos e anatomopatológicos dos prontuários de 71 doentes com adenocarcinoma gástrico submetidos à gastrectomia. O material obtido na operação foi submetido à análise imunoistoquímica e a frequência da expressão de cada proteína pôde ser analisada de acordo com a sua localização na célula e relacionada com as variáveis clinicopatológicas. Resultados: A graduação percentualda expressão e da localização das proteínas foi a seguinte: E-caderina em 3% na membrana; betacatenina em 23,4% no citoplasma e 3,1% no núcleo; APC em 94,6% no citoplasma; TCF-4 em19,4% no núcleo; e survivina em 93,9% no núcleo. Houve relação entre expressão da proteína E-caderina com a idade mais avançada (p=0,007); betacatenina com tumores <5 cm de diâmetro (p=0,041);APC com tumores proximais (p=0,047); e TCF-4 com tipo difuso da classificação de Lauren (p=0,017) e com o grau de penetração tumoral (p=0,002). Conclusão: A via Wnt/betacatenina não está envolvida na carcinogênese gástrica. Porém, a frequência elevada de survivina permite sugerir que outras vias sinalizadoras devam estar envolvidas na transformação do tecido gástrico.

Relationship of local recurrence with the expression of Survivin and MMP-2 in laryngeal carcinoma and its surgical margins / 临床耳鼻咽喉头颈外科杂志

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 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.

Study of the relationship among expression of Survivin and MRP and the drug resistance in human nasopharyngeal carcinoma / 临床耳鼻咽喉头颈外科杂志

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.

Expressions and correlations of Survivin, Ki67 and p53 in laryngeal squmous cell carcinoma / 临床耳鼻咽喉头颈外科杂志

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 0.05). 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.

Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells. RESULTS: The predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA. CONCLUSION: Silencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.

Synergistic tumor suppression by Ad. RGD-iNG4 in human nasopharyngeal carcinoma cell CNE and its mechanism / 临床耳鼻咽喉头颈外科杂志

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.

Combined application 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 / 临床耳鼻咽喉头颈外科杂志

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.

Avaliação de survivina e XIAP e dos fatores de transcrição foxo3a e foxM1 como potenciais biomarcadores de quimiorresistência no câncer de mama / [Evaluation of survivin and XIAP and transcription factors foxo3a and foxM1 as potential chemoresistance biomarkers in breast cancer]

O câncer de mama é a neoplasia que apresenta maior mortalidade entre as mulheres ao redor do mundo, apesar dos avanços na descoberta de novas modalidades terapêuticas e marcadores prognósticos. A resistência ao tratamento quimioterápico é uma das principais causas de falha terapêutica, apontando para a necessidade da identificação de biomarcadores preditivos de resposta. Evidências científicas mostram que a superexpressão de FoxM1 e das proteínas antiapoptóticas Survivina e XIAP, bem como a inativação do fator de transcrição Foxo3a, estão associados a quimioresistência e a um prognóstico desfavorável no câncer de mama. Dessa forma, o objetivo desse estudo é avaliar o papel das proteínas Survivina, XIAP, Foxo3a e FoxM1 como potenciais fatores de resistência à doxorrubicina (doxo), quimioterápico amplamente empregado no tratamento no câncer de mama. Nossos dados mostram que a doxo foi capaz de inibir a viabilidade celular nas linhagens celulares derivadas de carcinoma de mama MCF7 (não-invasiva, positiva para receptores de estrogênio e Her2) e MDA-MB-231 (invasiva, triplo-negativa), como avaliado pelo ensaio de MTT. A droga induziu a perda da adesão celular e fragmentação do DNA, como observado pela análise morfológica com quantificação das células não-aderidas e avaliação do conteúdo de DNA por citometria de fluxo. A análise da ativação das caspases-3, -7 e -9 por Western blotting revelou que a doxo induziu apoptose em células com diferentes status de p53. Em paralelo, o tratamento com a doxo resultou na redução dos níveis proteicos e de RNAm de XIAP e Survivina, como avaliado por Western blotting e PCR em tempo real, respectivamente. Entretanto, a indução da superexpressão da Survivina, por transfecção plasmidial, não foi capaz de conferir resistência ao quimioterápico. Corroborando esses resultados, observamos que o silenciamento gênico por siRNA de XIAP e Survivina, isoladamente ou em combinação, não sensibilizou as células à morte celular induzida pela doxo, indicando que tais proteínas não desempenham papel na resistência à droga. Contrariando dados da literatura, a doxo foi capaz de induzir a fosforilação de Foxo3a e Akt e reduzir a expressão de seu alvo transcricional Bim e dos níveis de RNAm de FOXO3A. De maneira consistente, a droga promoveu a translocação de Foxo3a do núcleo para o citoplasma, como examinado por fracionamento subcelular, apontando para a inativação da sua função. Além disso, a expressão do fator de transcrição FoxM1 foi reduzida, mediante o estímulo apoptótico induzido pela doxo. A indução da superexpressão de FoxM1 foi capaz de reverter a sensibilidade das células MDA-MB-231 à doxo, processo que envolveu a indução dos níveis de Survivina e XIAP. O mesmo efeito não foi observado nas células MCF7 superexpressando FoxM1, uma vez que se mantiveram sensíveis ao quimioterápico e apresentaram inalterados níveis de Survivina e XIAP. O conjunto dos nossos dados indica que a via de sinalização oncogênica mediada pelo fator de transcrição FoxM1 é capaz de promover a resistência à doxo e sugere que a combinação clínica de inibidores de FoxM1 com a doxo tem o potencial de sobrepujar a quimiorresistência no câncer de mama, principalmente em tumores triplo-negativos.

Breast cancer is the leading cause of deaths in women around the world, despite recent advances regarding novel therapeutic options and identification of prognostic factors. Resistance to therapy is the main cause of treatment failure and still there is no predictive biomarker for response to systemic therapy available. Increasing evidence shows that Survivin and XIAP antiapoptotic proteins and FoxM1 overexpression, as well as the inactivation of Foxo3a transcription factor, are closely associated with chemoresistance and poor prognosis in breast cancer. Thus, this study aimed to investigate Survivin, XIAP, Foxo3a and FoxM1 potential role on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer. Our data demonstrates that dox inhibited cell viability in the breast cancer derived cell lines MCF7 (non-invasive, Her2 and estrogen receptor positive) and MDA-MB- 231 (invasive, triple-negative), as evaluated through the MTT assay. The drug induced loss of cell adhesion and DNA fragmentation, as examined by morphological analysis followed by quantification of non-adherent cells and flow cytometry DNA content analysis. Western blotting evaluation of caspases-3, -7 and -9 activation revealed that dox induced apoptosis in cells with different p53 activation status. In parallel, exposure to dox resulted in reduction in Survivin and XIAP protein and mRNA levels, as evaluated by Western blotting and real time PCR, respectively. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. Accordingly, XIAP and Survivin silencing through siRNA, individually or in combination, had little effect on breast cancer cells sensitivity towards dox, suggesting that the drug can induce apoptosis independently of their expression. Contrasting data in the literature, dox treatment induced Foxo3a and Akt phosphorylation and reduced the expression of its transcriptional target Bim and FOXO3A mRNA levels. In agreement, dox-exposed cells displayed Foxo3a expression in cytoplasm, differently from predominantly nuclear Foxo3a observed in untreated cells, as examined through subcellular localization. These data point to dox-induced Foxo3a inactivation in breast cancer cells. In addition, we observed that FoxM1 transcription factor expression was inhibited upon dox-mediated apoptotic stimuli. Importantly, FoxM1 overexpression could counteract apoptosis in MDA-MB-231 cells, along with induction of Survivin and XIAP expression. This effect was not observed in MCF7 cells, which remained similarly sensitive to dox and displayed Survivin and XIAP levels unaltered. Altogether, our results demonstrate that FoxM1 signaling pathway can promote dox resistance and suggest that combining FoxM1 inhibitors with dox has the potential to circumvent chemoresistance in breast cancer, specially in triple negative tumors.

Expression of survivin in rheumatoid arthritis / 中南大学学报(医学版)

OBJECTIVE@#To detect the correlation between survivin and rheumatoid arthritis (RA) to determine the possible mechanism of RA and multidrug resistance in refractory rheumatoid arthritis (RRA).@*METHODS@#We collected 15 normal controls, 35 early untreated RA patients, 20 effectively treated RA patients and 25 RRA patients according to selection standard. The expression of survivin in the peripheral blood lymphocytes was detected by immunocytochemical method.@*RESULTS@#There was significant difference in the survivin expression in the peripheral blood lymphocytes between the early untreated and normal control group (χ(2)=29.59, P0.05). The survivin expression in the peripheral blood lymphocytes of the RRA group was significantly stronger than in the effectively treated RA group (χ(2)=24.35, P0.05).@*CONCLUSION@#Survivin has an influential role in the occurrence and development of rheumatism arthritis. Survivin might be involved in refractory multidrug resistance of RA and be one of the multidrug resistance mechanism of RRA.

Expression and significance of survivin and PCNA in sinonasal inverted papilloma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore the expression and significance of survivin and proliferating cell nuclear antigen (PCNA) on the occurrence, proliferation, recurrence and carcinogenesis of the sinonasal inverted papilloma (SNIP).@*METHOD@#Immunohistochemical method was used to detect the expression of survivin and PCNA in 10 cases of nasal cavity mucosal (NM), 45 cases of SNIP and 9 cases of canceration SNIP.@*RESULT@#The positive expression of survivin and PCNA increased gradually in NM,SNIP and canceration PCNA group, and there were significant difference between the three groups. But there was no correlation between survivin and PCNA in the tissue of SNIP (r = 0.135, P > 0.05).@*CONCLUSION@#Survivin and PCNA are involved in the growth and carcinogenesis of SNIP.

The expression of Smac and survivin in sinonasal inverted papilloma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To explore the expression of Smac(second mitochondria-derived activator of caspase)and survivin on the growth, development, recurrence and carcinogenesis of the sinonasal inverted papilloma(NIP).@*METHOD@#Immunohistochemical method was used to detective the expression of Smac and survivin in 10 cases of (nasal cavity mucosae, NM), 45 cases of NIP. The NIP group including 25 cases of NIP without dysplasia, 11 cases of NIP with dysplasia, and 9 cases of NIP with malignant transformation to squamous cell carcinoma(SCC).@*RESULT@#The intensity of the positive expression of Smac in NIP was lower than NM, the intensity of the positive expression decreased with the decreasing degree of histological differentiation. There was significantly difference between NIP without dysplasia and SCC. The expression of survivin was negative in the control group, the expression intensity of NIP was enhanced. The degree of histological differentiation was lower, the intensity of the positive expression was higher. The expression between NIP without dysplasia and SCC had significantly differences. Smac negatively correlated with survivin(rs = -0.403, P<0.01).@*CONCLUSION@#Smac and survivin were associated with the growth and carcinogenesis of NIP.

Effects of the inhibition and apoptosis by shRNA mediated survivin gene silencing in xenograft tumor of nasopharyngeal carcinoma cell CNE-2 / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe wether pSIREN-survivin/shRNA can induce the apoptosis and inhibit it's growth of nude mice xenograft tumor of nasopharyngeal carcinoma cell.@*METHOD@#Subcutaneous tumors in athymic mice were induced by inoculation of NPC and divided into the blank group A, the negative group B,the experimental group C randomly. PBS,the negative and positive pSIREN-survivin/shRNA were injected into the subcutaneous tumors poly site. The inhibition ratio was measured by the tumor size calculation after inoculation. The expression of survivin in the xenograft tumor was observed by RT-PCR and immunohistochemistry. The cell apoptosis in tumor tissues was detected by TEM. The liver and kidney function was tested by blood routine test.@*RESULT@#The inhibition ratio of group C and group B was (52. 11 +/- 1. 03)%, (2. 15 0. 11)% respectively, the inhibition rate in expression level of survivin mRNA was (77. 5+/-2. 03) %, (1. 39+/-0. 14) % respectively. The dyeing of survivin was more shallow in group C, the intensity is also less than group B. Nuclear chromatin was deepened, split into pieces, flake, and nuclear membrane was surrounded to edge. Cytoplasmic color and density were deepened, and organelles such as mitochondria was disappeared, the microscopic changes of cellular apoptosis at the earlier stage were watched, the difference of the function in liver and kidney was not statistically in statistical.@*CONCLUSION@#The expression of survivin in xenograft tumor was significantly inhibited by pshRNA-survivin/shRNA,the apoptosis of tumor cells was accelerated and the growth speed of NPC cells in xenograft tumor was retarded. The high expression of nasopharyngeal carcinoma's gene could significantly be silenced by using technology of RNAi, the growth of tumors could be inhibited also. Its a novel treatment that have a good prospect.

Simultaneous inhibition of XIAP and survivin expression on EMT and invasion of human pancreatic cancer cells / 中南大学学报(医学版)

OBJECTIVE@#To investigate the simultaneous inhibition of X-linked inhibitor of apoptosis protein (XIAP) and survivin expression on epithelial-mesenchymal transition (EMT) and invasiion of pancreatic cancer cells Panc-1, and its mechanism.@*METHODS@#On the established human pancreatic cancer cells Panc-1-XS, the expression of XIAP and survivin was inhibited simultaneously. Cell invasion and migration were detected by Transwell chamber experiments and scratch test, and the expression of epithelial marker E-cadherin, mesenchymal markers Slug, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and P-Akt protein was determined by Western blot.@*RESULTS@#Cell invasion and migration of Panc-1-XS cells decreased significantly, accompanied by significantly upregulated protein expression of E-cadherin, and significantly declined protein expression of the Slug, indicating increased mesenchymal-epithelial conversion (MET); and increased protein expression of PTEN, and declined protein expression of P-Akt.@*CONCLUSION@#Simultaneously inhibiting the expression of XIAP and survivin can partially reverse EMT phenotype of pancreatic cancer Panc-1 cells, which then significantly reduces the cell invasion and migration of Panc-1 cell lines. This process may be regulated by PTEN/PI3K/Akt signaling pathway.