Protein Expression Levels Of Survivin Research Articles

Co-Delivery of erlotinib and resveratrol via nanostructured lipid Carriers: A synergistically promising approach for cell proliferation prevention and ROS-Mediated apoptosis activation

Cancer treatments are always associated with various challenges, and scientists are constantly trying to find new therapies and methods. Erlotinib (ELT) is a well-known medicine against non-small cell lung cancer (NSCLC). However, treatments by ELT disrupt therapy due to drug resistance and pose severe challenges to patients. To achieve high-performance treatment, we gained nanostructured lipid carriers (NLCs) to evaluate synergistic anticancer effects of co-delivery of ELT and resveratrol (RES), a natural herbal derived phenol against NSCLC. NLCs are prepared via the hot homogenization method and characterized. In vitro cytotoxicity of formulations were evaluated on adenocarcinoma human alveolar basal epithelial (A549) cells. Prepared NLCs showed a narrow particle size (97.52 ± 17.14 nm), negative zeta potential (-7.67 ± 4.55 mV), and high encapsulation efficiency (EE%) was measured for the prepared co-delivery system (EE% 89.5 ± 5.16 % for ELT and 90.1 ± 6.61 % for RES). In vitro outcomes from cell viability study (12.63 % after 48 h of treatment), apoptosis assay (85.50%.), cell cycle (40.00% arrest in G2-M), and western blotting investigations (decreasing of protein expression levels of survivin, Bcl-2, P-Caspase 3P-caspase 9, and P-ERK 1/2, and additionally, increasing protein levels of BAX, P53, C-Caspase 3 and 9), DAPI staining, and colony formation assays showed the augment cytotoxic performances for co-delivery of ELT and RES loaded NLCs. Our study introduced the co-delivery of ELT and RES by NLCs as a novel strategy to elevate the efficacy of chemotherapeutics for NSCLC.

Paeonol enhances TRAIL-induced apoptosis of human lung cancer cells by upregulating death receptors-4 and 5 via ROS-JNK/ERK-CHOP signaling


 
 
 
 Purpose: To study the anti-proliferative potential of paeonol against lung cancer cells, and investigate its mechanism of action.
 Methods: Cell viability after paeonol treatment was determined with 3-(4,5-dimethylthiazol-2yl)2,5- diphenyltetrazolium bromide (MTT) assay, while paeonol- and TRAIL-mediated apoptosis was assayed using flow cytometry. Western blotting was used to assay the protein expression levels of phosphorylated JNK and ERK1/2, as well as protein expressions of pro-apoptotic factors/death receptors. 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) staining and flow cytometry were used to monitor paeonol-induced reactive oxygen species (ROS) in the cells.
 Results: Paeonol treatment markedly reduced the proliferations of H1975 and BGC823 cells (p < 0.05). In H1975 and BGC823 cells, paeonol/TRAIL combination increased apoptosis to 88.43 and 87.21 %, respectively (p < 0.05). The levels of death receptor 4 (DR4) and death receptor 5 (DR5) were increased significantly by paeonol, relative to the control (p < 0.05). Paeonol also reduced the levels of decoy receptor-1 (DcR1) and decoy receptor-2 (DcR2), and increased the expression of CHOP (p < 0.05). The protein expression levels of survivin, Bcl-2, cFLIP and Bcl-xL were decreased, while protein levels of caspase3, caspase-8 and caspase-9 were upregulated by paeonol. Moreover, paeonol significantly upregulated p-ERK and p-JNK in H1975 and BGC823 cells, and also increased ROS levels, when compared to control (p < 0.05).
 Conclusion: Paeonol exerts anti-proliferative potential on lung cancer cells through upregulation of death receptors, activation of JNK/ERK-CHOP pathway and generation of ROS. Therefore, paeonol has a therapeutic potential for the management of lung cancer.
 
 
 

The Establishment of Quantitatively Regulating Expression Cassette with sgRNA Targeting BIRC5 to Elucidate the Synergistic Pathway of Survivin with P-Glycoprotein in Cancer Multi-Drug Resistance.

Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.

Emodin Reverses Gemcitabine Resistance of Pancreatic Cancer Cell Lines Through Inhibition of IKKβ/NF-κB Signaling Pathway.

BackgroundPancreatic cancer is one of the most malignant tumors, and gemcitabine has been considered as the standard treatment and been widely utilized as a first-line drug for advanced pancreatic cancer, but gemcitabine-resistance always occurs after a short period of treatment.MethodsTwo pancreatic cancer cell lines Panc-1 and MIA-PaCa-2 were used as the study subject and their gemcitabine-resistant cells were established. Both drug-resistant cells were divided into four groups: blank, emodin, gemcitabine, and emodin+gemcitabine. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis rate and P-gp function. Quantitative real-time polymerase chain reaction and Western blotting were used to detect Survivin, XIAP, Caspase-9/3, NF-κB p65, IKKβ and IκB-α mRNA/protein expressions, respectively. Electrophoretic mobility shift assay (EMSA) was performed to detect NF-κB binding activity. Rhodamine 123 efflux assay was used to detect P-gp function.ResultsEmodin could inhibit cell activity in all cell lines. Both emodin and gemcitabine can significantly increase the apoptosis rate, and the combination of the two drugs can further significantly increase the apoptosis rate in normal pancreatic cancer cell lines. In both drug-resistant pancreatic cancer cell lines, it can be observed that although gemcitabine can increase the apoptosis rate, the effect of promoting apoptosis is significantly lower than that of emodin; the drug combination can still significantly increase the apoptosis rate on the basis of emodin alone. Emodin can significantly reduce the mRNA and protein expression levels of Survivin, XIAP, NF-κB, and IKKβ, and significantly increase the mRNA and protein expression levels of Caspase-3/9 and IκB-α. Emodin significantly reduced NF-κB activity and emodin significantly promoted P-gp fluorescence intensity from Rhodamine 123 efflux assay.ConclusionEmodin inhibits the expression of IKKβ, thereby inhibiting the expression and activity of downstream NF-κB, and inhibits P-gp function at the same time, ultimately achieving the purpose of reversing the drug-resistance of pancreatic cancer cell lines.

Upregulation of DR5 and Downregulation of Survivin by IITZ-01, Lysosomotropic Autophagy Inhibitor, Potentiates TRAIL-Mediated Apoptosis in Renal Cancer Cells via Ubiquitin-Proteasome Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively is able to increase apoptosis in cancer cells as agent with minimum toxicity to noncancerous cells. However, all cancer cells are not sensitive to TRAIL-induced apoptosis. In this study, we showed the sub-lethal concentrations of a lysosomotropic autophagy inhibitor, IITZ-01, sensitizes cancer cells (renal, lung, and breast carcinoma) to TRAIL-induced apoptosis through DR5 upregulation and survivin downregulation through ubiquitin-proteasome pathway. Knockdown of DR5 or overexpression of survivin inhibited combined treatment with IITZ-01 and TRAIL-induced apoptosis. IITZ-01 downregulated protein expression of Cbl, ubiquitin E3 ligase, and decreased expression level of Cbl markedly led to increase DR5 protein expression and TRAIL sensitivity. Moreover, IITZ-01 decreased expression level of survivin protein via downregulation of deubiquitinase ubiquitin-specific protease 9X (USP9X) expression. Taken together, these results provide the first evidence that IITZ-01 enhances TRAIL-mediated apoptosis through DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation in renal carcinoma cells.

Lung cancer A549 cells suppressed with overexpressed HNF1B or PCDHA13 inhibited PI3K/AKT phosphorylation.

BackgroundLung cancer is the most revenant and deadly tumors around the world. Here we aimed to explore the effects of hepatocyte nuclear factor 1B (HNF1B) and PCDHA13 overexpression on PI3K/AKT phosphorylation and malignant biological behavior in lung cancer A549 cells.MethodsHNF1B and PCDHA13 were amplified, and their overexpression plasmids were constructed for transfection. RT-PCR was used to detect the mRNA levels of HNF1B and PCDHA13. Cell proliferation and cell apoptosis were detected by clone formation experiments and flow cytometry, respectively, while cell invasion was studied by Transwell assay. The expression of survivin, PCNA, Caspase-3, Caspase-9, VEGF, and fibronectin was detected using immunoblotting, as was PI3K/AKT phosphorylation.ResultsThe level of HNF1B mRNA expression was significantly higher in the pcNDA-HNF1B group than in the control group (P<0.05), and the level of PCDHA13 mRNA expression in the pcNDA-PCDHA13 group was also significantly increased (P<0.05). The clone formation rate and cell invasion count in pcNDA-HNF1B or pcNDA-PCDHA13 transfected groups were significantly reduced in comparison with the control group, which were further validated with the protein expression levels of survivin, PCNA, VEGF, and fibronectin (P<0.05). However, the apoptosis rate, and the cleaved caspase3/caspase3 and cleaved caspase9/caspase9 protein expression ratios were all significantly increased (P<0.05). Cells transfected with pcNDA-HNF1B or pcNDA-PCDHA13 showed decreased levels of PI3K/AKT phosphorylation (P<0.05).ConclusionsOverexpression of HNF1B and PCDHA13 inhibits the phosphorylation of PI3K/AKT and hinders the malignant biological behavior of lung cancer A549 cells.

Expression of anti-apoptotic protein survivin in human endometrial carcinoma: Clinical and pathological associations as a separate factor and in combination with concomitant PTEN and p53 expression.

Endometrial carcinoma is one of the most common types of gynecological cancer. A total of 99 cases of primary endometrial carcinoma were investigated for survivin expression by immunohistochemistry. Furthermore, the association between concomitant survivin, PTEN and p53 expression, and clinicopathological parameters was examined. Immunopositivity for survivin was identified in 88% of cases. Concomitant survivin, PTEN and p53 expression (staining scores and intensity) was observed in 60% of endometrial adenocarcinomas. A significant association was identified between the sum of staining intensity and scores of survivin immunopositive cells, and patient age (P=0.028), histological grade (P<0.001), clinical stage (P=0.018) and fallopian tube and/or ovarian invasion (P=0.039). A negative tendency for correlation was observed between surivin and PTEN immunostaining scores (P=0.062; ρ=−0.238). Specimens with high scores of survivin expression tended to show decreased scores of PTEN immunostaining, and vice versa. However, in circumstances with an increased co-expression of survivin and PTEN, a statistically significant association with histological types was observed (P=0.020). A statistically significant positive correlation was identified between survivin and p53 sum co-expression (P=0.008; ρ=0.300). Furthermore, a significant association was identified between survivin and p53 concomitant sum expression and age of patients (P=0.001), histological type (P=0.020), clinical stage (P=0.037), histological differentiation (P=0.001) and presence of fallopian tube and/or ovarian invasion (P=0.026). The present findings suggested that survivin may be an indicator of unfavorable outcome in older patients with endometrial carcinoma, in specific circumstances that are dependent on different concomitant genetic alterations and different combinations of molecular signaling pathways. Increased expression levels of survivin and PTEN may serve a role in the development of more aggressive endometrial carcinoma during their interaction. In addition, protein expression levels of survivin and p53 are positively correlated and may share a common molecular pathway to promote endometrial carcinogenesis. These findings provided evidence that survivin and p53 combined may be useful markers for the prediction of tumor behavior and prognosis.

Discovery of inner centromere protein-derived small peptides for cancer imaging and treatment targeting survivin.

Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer‐specific treatment. In this study, we designed and synthesized 7‐19 residues of inner centromere protein (INCENP)‐derived small peptides (INC peptides) as novel survivin‐targeting agents. The INC peptides showed binding affinity for the human survivin protein (K d = 91.4‐255 nmol L−1); INC16‐22, which contains residues 16‐22 of INCENP, showed the highest affinity (91.4 nmol L−1). Confocal fluorescence imaging showed consistent colocalization of FITC‐INC16‐22 and survivin in cell lines. Nona‐arginine‐linked INC16‐22 (r9‐INC16‐22) rendered INC16‐22 cells penetrable and strongly inhibited cell growth of MIA PaCa‐2 cells (52% inhibition at 1.0 µmol L−1) and MDA‐MB‐231 cells (60% inhibition at 10 µmol L−1) as determined by MTT assays. The exposure of MIA PaCa‐2 cells to 40 µmol L−1 r9‐INC16‐22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase‐3 was significantly increased in cells treated with r9‐INC16‐22, even at 10 µmol L−1, compared to untreated cells. Flow cytometry revealed that r9‐INC16‐22 strongly induced apoptosis in MIA PaCa‐2 cells. These results indicate that the cytotoxic effects of r9‐INC16‐22 could be mediated mainly through the disruption of survivin‐dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.

Survivin expression modulates the sensitivity of A549 lung cancer cells resistance to vincristine.

Lung cancer is the leading cause of cancer-associated mortalities worldwide. Chemotherapeutic drug vincristine is widely used to treat lung cancer; however, the acquisition of drug resistance is the major limitation of chemotherapy, and it is thus important to determine the mechanism underlying vincristine resistance in lung cancer. Survivin has been reported to be associated with the development of drug resistance and be involved in the progression of non-small cell lung cancer. In the present study, a vincristine-resistant lung cancer cell line, A549/VCR, was used to investigate the possible involvement of survivin in the acquisition of vincristine resistance. Western blot analysis demonstrated that survivin protein expression level was markedly higher in A549/VCR cells compared with in control A549 cells, whereas p53 expression level was lower in A549/VCR cells compared with in A549 cells. Thus, wild-type p53 was overexpressed in A549/VCR cells and it reversed vincristine resistance of A549/VCR cells via the inhibition of survivin expression. Furthermore, survivin was knocked down by small interfering RNA technology and the effects on viability and apoptosis of resistant cells were investigated. MTT, Annexin V-fluorescein isothiocyanate/propidium iodide and caspase-3 activity assays indicated that survivin silencing significantly inhibited cell viability and enhanced apoptosis induced by vincristine treatment in A549/VCR cells compared with non-silenced A549/VCR cells. These results suggested that survivin expression regulated by p53 may serve an important role in drug resistance in A549/VCR cells and may be a potential target for enhancing vincristine sensitivity in A549 lung cancer cells. Additionally, the present study revealed that A549/VCR cells exhibited cross resistance to methotrexate (MTX) and survivin silencing re-sensitized A549/VCR cells to MTX, indicating the crucial role of survivin in regulating A549 cells sensitivity to anticancer drugs. The results of the present study are significant for determining the underlying mechanism of vincristine resistance in lung cancer.

Endocrine therapy inhibits proliferation and migration, promotes apoptosis and suppresses survivin protein expression in colorectal cancer cells.

The majority of colorectal cancers (CRCs) are hormone-dependent. Thus, endocrine therapy has become an attractive strategy to treat CRC. The aim of the present study was to investigate the inhibitory effect of combined tamoxifen (TAM) plus β-estradiol (E2) treatment on human DLD-1 CRC cells. The human DLD-1 CRC cell line was treated with different concentrations of TAM, β-estradiol, or a combination of these two agents. Cell viability was assessed using an MTT assay, while apoptosis was detected using flow cytometry analysis. Alterations in the RNA and protein levels of the apoptosis-associated factors cyclin D1 and survivin were measured in the treated DLD-1 cells using semi-quantitative polymerase chain reaction (sqPCR) and western blot analyses. Alterations in cellular migration ability were monitored using a Transwell migration assay. Treatment with TAM, β-estradiol and TAM plus β-estradiol inhibited DLD-1 cell viability. The flow cytometry results revealed that these drugs promoted cell apoptosis, and the Transwell migration assay results indicated that the reduction in cell migration was greater in the TAM+E2 treatment group when compared with each treatment alone. sqPCR and western blot analysis results demonstrated that TAM, E2 and a combination of the two affected survivin expression based on the drug concentration and the treatment duration; however, they demonstrated no significant effect on cyclin D1 expression. In conclusion, treatment of DLD-1 cells with TAM, β-estradiol, or a combination of these two drugs, inhibited cell viability and migration, promoted cell apoptosis, and reduced the mRNA and protein expression levels of survivin in a dose- and time-dependent manner. These results provide novel experimental basis for hormonal adjuvant therapy for the treatment of CRC.

Preclinical assesement of survivin and XIAP as prognostic biomarkers and therapeutic targets in gastroenteropancreatic neuroendocrine neoplasia

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) represent a rare and heterogenous tumor entity. Importantly, the highly proliferative subgroup of neuroendocrine carcinoma (GEP-NEC) is characterized by high resistance to conventional chemotherapy. Consequently, there is an urgent need to identify novel therapeutic targets, especially for GEP-NEC. Thus, we focused on Inhibitor of apoptosis protein (IAP) family members survivin and XIAP that orchestrate inhibition of apoptosis, induce resistance against chemotherapeutics and facilitate tumor metastasis. Copy number gains (CNGs) could be detected by microarray comparative genomic hybridization for survivin and XIAP in 60 % and 26.7 % of all GEP-NENs, respectively. Immunohistochemical staining of tissue specimens from 77 consecutive patients with GEP-NEN demonstrated increased survivin protein expression levels in tissue specimens of highly proliferative GEP-NEC or GEP-NEN located in the stomach and colon. In contrast, XIAP overexpression was associated with advanced tumor stages. Knockdown of survivin and XIAP markedly reduced cell proliferation and tumor growth. In vitro, YM155 induced apoptotic cell death accompanied by a reduction in cell proliferation and inhibited GEP-NEC xenograft growth. Taken together, our data provide evidence for a biological relevance of these IAPs in GEP-NEN and support a potential role of survivin as therapeutic target especially in the subgroup of aggressive GEP-NEC.

Curcumin reduces the expression of survivin, leading to enhancement of arsenic trioxide-induced apoptosis in myelodysplastic syndrome and leukemia stem-like cells.

Low response, treatment-related complications and relapse due to the low sensitivity of myelodysplastic syndrome (MDS) and leukemia stem cells (LSCs) or pre-LSCs to arsenic trioxide (ATO), represent the main problems following treatment with ATO alone in patients with MDS. To solve these problems, a chemosensitization agent can be applied to increase the susceptibility of these cells to ATO. Curcumin (CUR), which possesses a wide range of anticancer activities, is a commonly used chemosensitization agent for various types of tumors, including hematopoietic malignancies. In the present study, we investigated the cytotoxic effects and potential mechanisms in MDS-SKM-1 and leukemia stem-like KG1a cells treated with CUR and ATO alone or in combination. CUR and ATO exhibited growth inhibition detected by MTT assays and apoptosis analyzed by Annexin V/PI analyses in both SKM-1 and KG1a cells. Apoptosis of SKM-1 and KG1a cells determined by Annexin V/PI was significantly enhanced in the combination groups compared with the groups treated with either agent alone. Further evaluation was performed by western blotting for two hallmark markers of apoptosis, caspase-3 and cleaved-PARP. Co-treatment of the cells with CUR and ATO resulted in significant synergistic effects. In SKM-1 and KG1a cells, 31 and 13 proteins analyzed by protein array assays were modulated, respectively. Notably, survivin protein expression levels were downregulated in both cell lines treated with CUR alone and in combination with ATO, particularly in the latter case. Susceptibility to apoptosis was significantly increased in SKM-1 and KG1a cells treated with siRNA-survivin and ATO. These results suggested that CUR increased the sensitivity of SKM-1 and KG1a cells to ATO by downregulating the expression of survivin.

Effects of survivin on FVADT chemotherapy for refractory multiple myeloma.

The present study aimed to investigate the effects of survivin, an apoptosis inhibitor protein, on the efficacy of the fludarabine, vincristine, epirubicin, dexamethasone and thalidomide (FVADT) chemotherapy regime for the treatment of refractory multiple myeloma (MM). A total of 82 patients with MM were selected from the Hematology Inpatient Department at The Second Affiliated Hospital of Zhengzhou University (Zhengzhou, China). The initial treatment group consisted of 40 patients with MM, who received the vincristine, epirubicin and dexamethasone (VAD) chemotherapy regime. The refractory group consisted of 42 patients with refractory MM, who received the FVADT chemotherapy regime. Bone marrow biopsies were collected via marrow aspirations, and the protein expression of survivin was analyzed by immunohistochemistry. In addition, the Kaplan-Meier method was used for survival analyses. Intergroup differences in the protein expression levels of survivin were compared, and the association between survivin expression and the short- and long-term effects of FVADT chemotherapy were analyzed. The positive expression rate of survivin was significantly higher in the refractory group, as compared with the initial treatment group (P<0.05). Furthermore, the complete remission rate and the effective rate were significantly lower in the survivin-positive group, as compared with the survivin-negative group (P<0.05). The overall survival, progression free survival and 1 and 3 year survival rates of the survivin-positive group were significantly higher, as compared with the survivin-negative group (P<0.05). The results of the present study suggested that the protein expression of survivin was upregulated in refractory MM tissues, which was indicative of a poor short- and long-term efficacy for FVADT chemotherapy.

Effects of LG268 on Cell Proliferation and Apoptosis of NB4 Cells.

Aims: To investigate the effect of LG100268 (LG268) on cell proliferation and apoptosis in NB4 cells.Methods: NB4 cells were treated with LG268 for 24 h or 48 h. The effect of LG268 on cell proliferation was assessed by the CCK-8 assay and colony-forming assay. Apoptosis and cell cycle were evaluated by flow cytometry. The protein expression levels of Survivin, PARP, c-Myc, cyclin D1, ERK, p-ERK, p38 MAPK, and p- p38 MAPK were detected by western blot.Results: We found that LG268 inhibited the proliferation of NB4 cells in a dose-dependent manner. Flow cytometry analysis showed that LG268 accelerated apoptosis in NB4 cells in a time- dependent manner and that LG268 treatment led to cell cycle arrest at G0/G1 phase. Moreover, LG268 significantly decreased the protein levels of Survivin, c-Myc, and cyclinD1. Cleaved PARP was observed in the LG268 treatment group but not in the control group. In addition, LG268 increased the phosphorylation level of p38 MAPK and decreased the phosphorylation level of ERK.Conclusions: LG268 inhibited cell proliferation and promoted cell apoptosis in NB4 cells.

MicroRNA-542-3p suppresses cellular proliferation of bladder cancer cells through post-transcriptionally regulating survivin

AimTo investigate the clinical significance of microRNA-542-3p (miR-542-3p) and its target gene survivin in human bladder cancer, and to determine their functions in malignant phenotypes of this disease. MethodsExpression levels of miR-542-3p and survivin mRNA in bladder cancer and adjacent normal tissues were detected by quantitative RT-PCR. Cell proliferation and cell cycle were assessed by cell viability assay, flow cytometry and colony formation assay based on two human bladder cancer cell lines. ResultsMiR-542-3p expression was downregulated, while survivin mRNA expression was upregulated in bladder cancer tissues, compared to adjacent normal tissues (both P<0.001). Importantly, the expression level of miR-542-3p in bladder cancer tissues was negatively correlated with that of survivin mRNA. MiR-542-3p-low and/or survivin-high expression were all significantly associated with tumor stage (all P<0.05) and tumor recurrence (all P<0.05) of patients with bladder cancer. Moreover, the enforced expression of miR-542-3p remarkably inhibited cell proliferation by inducing G1 phase arrest in both T24 and J82 cells, and decreased the expression level of survivin protein. In contrast, the knock-down of miR-542-3p dramatically promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression level of survivin protein. ConclusionMiR-542-3p and its target gene survivin may play crucial roles in the aggressive progression of human bladder cancer. More interestingly, miR-542-3p may function as a tumor suppressor and inhibit the proliferation of bladder cancer cells, implying that miR-542-3p-survivin signal axis might be a novel therapeutic target of this disease.

S6K1 inhibition enhances the apoptotic cell death of breast cancer cells in response to Bcl-2/Bcl-xL inhibition by the downregulation of survivin.

Breast cancer cells possess a deregulated apoptotic pathway with increased expression levels of anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins and ribosomal S6 kinase 1 (S6K1) protein activity. Therefore, combined interference of anti-apoptotic Bcl-2 family and S6K1 protein expression may be a reasonable therapeutic strategy for the treatment of patients with breast cancer. In the present study, it was identified that the administration of a combination of ABT263 [navitoclax; a Bcl-2/Bcl-extra large (Bcl-xL) inhibitor] and PF4708671 (an S6K1 inhibitor) markedly increased apoptotic cell death in the BT474 breast cancer cells compared with the administration of either agent alone. Furthermore, the downregulation of Bcl-2/Bcl-xL and S6K1 with small interfering RNA induced a significant increase in cell death compared with RNA interference of either agent alone. Notably, combination treatment with ABT263 and PF4708671 decreased the expression level of survivin protein, with this ectopic expression of survivin attenuating cell death. Thus, the present study determined that the combined inhibition of Bcl-2/Bcl-xL and S6K1 may be a good strategy for treating patients with breast cancer.

Effect of quercetin on the proliferation of the human ovarian cancer cell line SKOV-3 in vitro.

Quercetin is a hydrophobic agent that demonstrates potential anticancer activity. The aim of the present study was to observe the effects of quercetin on the proliferation and apoptosis of the ovarian cancer cell line SKOV-3, and to provide a foundation for the treatment of ovarian cancer using this agent. Ovarian cancer SKOV-3 cells were treated with quercetin at different doses. The inhibitory effect of quercetin on proliferation was detected using the MTT assay and the inhibition rate was calculated. Cell apoptosis was determined using Hoechst staining, and western blot analysis was used to analyze changes in the expression levels of survivin protein. The effects of quercetin on the cell cycle and apoptosis of the SKOV-3 cell line were analyzed using flow cytometry. Quercetin inhibited the proliferation of SKOV-3 cells in a time- and dose-dependent manner. Furthermore, Hoechst staining showed that quercetin induced SKOV-3 cell apoptosis. The protein expression levels of survivin were reduced as the concentration of quercetin increased. Flow cytometric analysis showed that quercetin caused ovarian cancer SKOV-3 cell cycle arrest in the G0/G1 phase and a significant decrease in the percentage of cells at the G2/M phase; furthermore, the apoptosis rate was observed to increase following quercetin treatment. The results in combination indicated that Quercetin could inhibit the proliferation of ovarian cancer SKOV-3 cells, inhibit cell cycle progression from G0/G1 to G2/M and induce cell apoptosis in vitro.

肝細胞癌細胞株MHCC97Hのサバイビン発現,増殖およびアポトーシスに及ぼす低分子干渉RNAの効果【Powered by NICT】

Objective To observe the effect of small interfering RNA(siRNA)targeting Survivin gene on urviving expression, proliferation and apoptosis of hepatocellular carcinoma cell line MHCC-97H. Methods Survivin sequence specific siRNA was designed and synthesized.siRNA/liposome complex was transfected into hepatocellular carcinoma cell line MHCC- 97H.The MHCC-97H cells were divided into Survivin siRNA group(Si-survivin), negative control siRNA group(NC group)and blank group(normal control group). Survivin mRNA and protein expressions were detected by reverse transcription-PCR and Western blot, respectively.The proliferation of MHCC-97H was measured by methythiazolydiphenyl-tetrazolium bromide assay.The Annexin V/PI double labeled flow cytometry was employed to measure the apoptosis at 24 h after transfection in different concentrations of Survivin siRNA(12.5, 25.0 and 50.0 nmol/L, respectively). Results After 48 h of transfection, the Survivin mRNA levels were 0.55±0.16(Si-survivin group), 0.85±0.28(NC group)and 0.93±0.40(normal control group), respectively, which were significantly different among 3 groups(F=414, P<0.01). The level of Survivin mRNA was the lowest in Si-survivin group, which was statistically different with NC group and normal control group(t=-20.56, -28.37, all P<0.001). The levels of Survivin protein expression in 3 groups were 0.602±0.005 (Si-survivin group), 0.835±0.007(NC group)and 0.993±0.003(normal control group)at 48 h after transfection, which were statistically different among 3 groups(F=238, P<0.01). The lowest level of protein expression was in Si-survivin group, which was statistically different with NC group and normal control group(t=-40.17, -66.03, all P<0.001). After 72 h and 96 h of transfection, the inhibitory rate of cell growth was significantly higher in Si-survivin group[(19.5±3.6)%, (12.0±0.9)%] compared with that in NC group[(3.6±0.9)%, (-1.3±6.1)%](t=36.18, 42.53, all P<0.05). The apoptosis rates in 12.5, 25.0, 50.0 nmol/L Survivin siRNA were (22.64±2.54)%, (35.37±3.28)% and (53.28±4.35)%, respectively.However, in NC group and normal control group, the apoptosis rates were (8.77±1.25)% and (9.72±1.37)%.The rates were statistically different among those 5 groups(F=35.93, P<0.01). And in the apoptosis rates of siRNA groups in different concentrations were statistically different when compared between each two groups(t=-29.73, -38.57, all P<0.001). Conclusion Survivin specific siRNA can inhibit the proliferation and induce the apoptosis by blocking Survivin gene expression in hepatocellular carcinoma cell line MHCC-97H. Key words: Hepatocellular carcinoma; Survivin; Small interfering RNA

Expression of survivin in adenoid cystic carcinoma of the lacrimal gland and the effect of intervention with arsenic trioxide in vitro.

The aim of the present study was to investigate the role of survivin in adenoid cystic carcinoma of the lacrimal gland (LGACC) and the effect of arsenic trioxide (As2O3) intervention in vitro. In total, 13 patients with LGACC were recruited from Renmin Hospital of Wuhan University between 2007 and 2012. The patients were divided into different groups, namely tumor-node-metastasis (TNM)1-2 and TNM3-4, according to the TNM classification. A total of eight samples were included in the TNM1-2 group, while five samples were included in the TNM3-4 group. In addition, six patients that suffered from other diseases (no tumor), but underwent eye surgery, were selected as the controls. The mRNA and protein expression levels of survivin were analyzed. In addition, primary adenoid cystic carcinoma (ACC) cells were cultured and the expression of survivin was silenced using short interfering RNA (siRNA), after which the cell proliferation was determined. Furthermore, the primary cultured ACC cells were treated with different doses of As2O3 to observe the effect on the apoptosis rate. The mRNA and protein expression levels of survivin in the LGACC groups were higher when compared with the controls (P<0.01). In addition, the expression levels were higher in the TNM3-4 group when compared with the TNM1-2 group (P<0.01). After silencing survivin expression using siRNA, the rate of ACC cell proliferation was shown to decrease at days 2, 3, 4 and 5 following culture, particularly in the TNM3-4 group at days 2 and 3 (P<0.05), day 4 (P<0.01 vs. TNM1-2 + siRNA); and days 3 and 5 (P<0.05), and day 2 (P<0.01 vs. TNM3-4 + siRNA). The mRNA expression levels of survivin in the TNM1-2 and TNM3-4 groups were significantly decreased following treatment with the various doses of As2O3 (2, 4 and 6 µΜ) for 48 h, and the apoptosis rate of the ACC cells was markedly increased (TNM1-2 and 3-4: As2O3 6 µM vs. As2O3 2 µM, As2O3 6 µM vs. As2O3 4 Μm, P<0.01; TNF3-4, As2O3 4 µM VS. As2O3 2 µM, P<0.01; TNM1-2, As2O3 4 µM vs. As2O3 2 µM, P<0.05). Therefore, survivin was demonstrated to be highly expressed in ACC cells of the lacrimal gland, and inhibition of survivin gene expression was found to suppress the proliferation of the ACC cells. Furthermore, As2O3 treatment was demonstrated to inhibit the mRNA expression of survivin in the ACC cells, while significantly increasing the apoptosis rate.

Combined therapeutic effect and molecular mechanisms of metformin and cisplatin in human lung cancer xenografts in nude mice.

This work was aimed at studying the inhibitory activity of metformin combined with the commonly used chemotherapy drug cisplatin in human lung cancer xenografts in nude mice. We also examined the combined effects of these drugs on the molecular expression of survivin, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor-C (VEGF-C), and vascular endothelial growth factor receptor-3 (VEGFR-3) to determine the mechanism of action and to explore the potential applications of the new effective drug therapy in lung cancer. The nude mice model of lung cancer xenografts was established, and mice were randomly divided into the metformin group, the cisplatin group, the metformin + cisplatin group, and the control group. The animals were killed 42 days after drug administration, and the tumor tissues were then sampled to detect the messenger ribonucleic acid (mRNA) and protein expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the cisplatin group and the combined treatment group were lower than that in the control group (P < 0.05). In the metformin group, the expression of MMP-2 protein and mRNA was lower than that in the control group (P < 0.05). The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the combined treatment group were lower than that in the cisplatin group and the metformin group (P < 0.05). Metformin inhibited the expression of MMP-2, cisplatin and the combined treatment inhibited the expression of survivin, MMP-2, VEGF-C, and VEGFR-3, and the combined treatment of metformin with cisplatin resulted in enhanced anti-tumor efficacy.