Survivin mRNA Research Articles

Small interfering RNA targeting of the survivin gene inhibits human tumor cell growth in vitro.

The present study aimed to evaluate the impact of small interfering RNA (siRNA) targeting of the survivin gene in human tumor cells and the effect of decreased survivin expression on the proliferation and apoptosis of tumor cells. Human tumor cell lines (MSA-MB-231, SGC-7901, HeLa, A549, SK-OV-3 and Raji, PC-3) were cultured in vitro and divided into three groups: survivin siRNA-treated, scrambled negative control siRNA-treated and an untreated control group. The level of survivin mRNA and protein expression was subsequently determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Cell proliferation was also examined by an MTT assay following transfection and the apoptotic rate of cells was detected by Hoechst and Annexin V/propidium iodide staining. It was observed that relative to the control group, expression of survivin mRNA and protein in the survivin siRNA-treated group was significantly downregulated. Furthermore, siRNA targeting of survivin lead to the inhibition of tumor cell proliferation, as well as an increase in their apoptotic rate in vitro. These data suggest that survivin may be a potential tumor biomarker and a novel target for the treatment of cancer.

CRA(Crosolic Acid) isolated from Actinidia valvata Dunn.Radix induces apoptosis of human gastric cancer cell line BGC823 in vitro via down-regulation of the NF-κB pathway

A natural ursolic compound, 2α,3β-dihydroxy-urs-12-en-28-oic acid (corosolic acid, CRA) was isolated from the root of Actinidia valvata Dunn. (A. valvata Radix). Since a large number of triterpenoid compound has marked anticancer effects toward various types of cancer cell lines in vitro, this study was carried out to investigate the anticancer effect of CRA in human gastric cancer cell line BGC823 cells and the underlying apoptotic mechanism of CRA was examined in BGC823 cell lines. The results showed that CRA significantly suppressed the viability of BGC823 cells in a concentration- and time-dependent manner. CRA also significantly increased the sub G1 population by cell cycle analysis in a concentration dependent manner. Exposure to CRA decreased p65, bcl-2, Fas, smac mRNA and protein expression, and increased IκBα, bax, survivin mRNA and protein expression. Results of immunofluorescence staining and EMSA further indicated CRA induced apoptosis by inhibiting nuclear translocation of nuclear factor NF-κB subunit p65. Consistently overall, our findings suggest that CRA induces apoptosis via inhibition of NF-κB (p65) expression level and activation of IκBα in BGC cells as a potent anticancer candidate for gastric cancer treatment.

Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells

Delivery of small interfering RNAs (siRNAs) into cells still remains a challenge in gene delivery studies. Here, we investigated the ability of synthesized Fe3O4-PEG-LAC-chitosan-PEI nanoparticles for siRNA delivery of survivin as the model gene into cells. The cellular uptake of survivin siRNA carried by synthesized nanoparticles into MCF-7 breast cancer cell line was evaluated by florescent microscopy and flowcytometry, both proving the efficacy of nanoparticles in delivery of up to 64.7% in comparison with lipofectamine 2000. Furthermore, the delivery of survivin siRNA by the nanoparticles (nanoplex) induced apoptosis that was assessed through DAPI staining and Annexin V/PI assays. In addition, we evaluated the efficacy of treatment with nanoplexes in the presence of mitoxantrone, as a chemotherapeutic agent. Our data indicated that inhibition of survivin expression increased the cell sensitivity to mitoxantrone. Real-time PCR and western blotting analysis revealed a significant reduction in mRNA and protein levels of survivin upon delivery of siRNA. Molecular docking studies showed that nanoparticles can bind to centeral BIR domain of survivin, exactly above zinc ion location with high affinity (ΔG: −10.3Kcal/mol). Also, thermodynamic studies proved the experimental results theoretically, revealing that the siRNA-loaded nanoparticles have a suppressing effect on survivin mRNA. Therefore, delivery of survivin siRNA into MCF-7 cells using Fe3O4-PEG-LAC-chitosan-PEI nanoparticles as a carrier enhances the cell death.

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.

Anticancer effects of β-elemene with hyperthermia in lung cancer cells.

β-elemene is a novel, plant-derived anticancer drug, which has been used to target multiple solid tumor types. Hyperthermia is an adjuvant therapeutic modality to treat cancer. However, the underlying mechanisms associated with the efficacy of these two treatments are largely unknown. The aim of the present study was to evaluate the effects of β-elemene combined with hyperthermia in lung cancer cell lines. An MTT assay was used to determine cell viability. The cell cycle and apoptosis were analyzed using flow cytometry. The morphology of cells during apoptosis was determined using a transmission electron microscope. The expression levels of P21, survivin, caspase-9, B-cell lymphoma 2 (Bcl-2) and Bcl-2-like protein 4 (Bax) mRNA were detected using quantitative polymerase chain reaction. β-elemene with hyperthermia treatment significantly inhibited the viability and increased the apoptosis rate of A549 cells compared with β-elemene treatment alone (P<0.01), and significantly decreased the proportion of cells in S phase compared with the control (P<0.01). Morphological observation using transmission electron microscopy indicated cross-sectional features of apoptosis: Chromatin condensation, reduced integrity of the plasma membrane, increased cellular granularity, nuclear collapse and the formation of apoptotic bodies. β-elemene with hyperthermia treatment significantly promoted P21 and Bax mRNA expression (P<0.01) and significantly decreased caspase-9, Bcl-2 and survivin mRNA expression (P<0.01) in A549 cells. In conclusion, β-elemene with hyperthermia has a significant inhibitory effect on A549 cells. This occurs through reducing S phase and inducing apoptosis, via an increase in P21 and Bax expression and a decrease in caspase-9, Bcl-2 and survivin expression.

Oxymatrine inhibits proliferation of human bladder cancer T24 cells by inducing apoptosis and cell cycle arrest

Oxymatrine has been shown to exert an antitumor effect on several types of cancer cells. However, the role of oxymatrine in bladder cancer has not yet been evaluated. The present study was designed to investigate the potential anti-proliferative effect of oxymatrine on bladder cancer T24 cells and the possible mechanisms involved. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine cell growth, and the cell morphology was examined using hematoxylin and eosin staining, wrights' staining and electron microscopy. The caspase-3 and survivin mRNA and protein levels were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The expression of tumor protein p53 (p53), Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) were analyzed using immunohistochemistry. Oxymatrine inhibited the proliferation of the T24 cells in a dose- and time-dependent manner. Oxymatrine also induced apoptosis and cell cycle arrest in the cells, in association with the upregulation of caspase-3 and Bax, and the downregulation of survivin, Bcl-2 and p53 expression. Overall, oxymatrine inhibits the proliferation of human bladder cancer cells by inducing apoptosis and cell cycle arrest via mechanisms that involve p53-Bax signaling and the downregulation of survivin expression.

Inhibition of bone formation in rats by aluminum exposure via Wnt/β-catenin pathway

The previous research found that aluminum trichloride (AlCl3) inhibited rat osteoblastic differentiation through inactivation of Wnt/β-catenin signaling pathway in vitro. On that basis, the experiment in vivo was conducted in this study. Rats were orally exposed to 0 (control group) and 0.4 g/L AlCl3 (AlCl3-treated group) for 30, 60, 90 or 120 days, respectively. We found that mRNA expressions of type I collagen and insulin-like growth factor-1, mRNA and protein expressions of Runx2 and survivin, ratio of p-GSK3β/GSK3β and protein expression of β-catenin were all decreased, whereas the mRNA and protein expressions Dkk1 and sFRP1 and the mRNA expressions and activity of Caspase-3 were increased in the AlCl3-treated group compared with the control group with time prolonged. These results suggest that AlCl3 inhibits bone formation and induces bone impairment by inhibiting the Wnt/β-catenin signaling pathway in young growing rats.

Gambogic acid potentiates the chemosensitivity of colorectal cancer cells to 5-fluorouracil by inhibiting proliferation and inducing apoptosis.

Chemotherapy using 5-fluorouracil (5-FU) for colorectal cancer (CRC) has low specificity and response rates, leading to severe side effects. Gambogic acid (GA), a traditional Chinese medicine, has multi-targeted anticancer effects, including growth inhibition and apoptosis induction. However, it is unclear whether a combination of 5-FU and GA has synergistic anticancer effects in CRC cells. In this study, SW480 and HCT116 human CRC cells and human intestinal epithelial cells (IECs) were treated with different concentrations of 5-FU, GA or 5-FU+GA. A Cell Counting kit-8 assay was conducted to quantify cell proliferation. The combination index (CI) was calculated and the median-effect principle was applied to analyze the interaction between 5-FU and GA. Flow cytometry was used to determine the percentage of cells undergoing apoptosis. Reverse transcription-quantitative polymerase chain reaction and western blotting were applied to measure P53, survivin and thymidylate synthase (TS) mRNA and protein levels. It was found that 5-FU+GA more pronouncedly inhibited cell growth and induced apoptosis, compared with either monotherapy. CI values <1 indicated the synergistic effects of the drugs. 5-FU+GA further decreased P53, survivin and TS mRNA and protein levels in the two CRC cell lines compared with single drugs, whereas increased P53 protein levels were observed in HCT116 cells. Moreover, 5-FU+GA did not increase cytotoxicity to IECs. These results demonstrate that GA enhances the anticancer effects of 5-FU on CRC cells. Combined treatment with 5-FU and GA is effective and safe for CRC cells, and may become a promising chemotherapy treatment.

Lymphotoxin β receptor activation promotes mRNA expression of RelA and pro-inflammatory cytokines TNFα and IL-1β in bladder cancer cells.

The role of inflammation in tumorigenesis and development is currently well established. Lymphotoxinβ receptor (LTβR) activation induces canonical and noncanonical nuclear factor (NF)‑κB signaling pathways, which are linked to inflammation‑induced carcinogenesis. In the present study, 5,637 bladder cancer cells were cultured and the activation of LTβR was induced by functional ligand, lymphotoxin (LT)α1β2, and silencing with shRNA. Reverse transcription‑quantitative polymerase chain reaction was utilized to detect the mRNA expression levels of NF‑κB family members RelA and RelB, cytokines including LTα, LTβ, tumor necrosis factor (TNF)α, TNF superfamily member 14, interleukin (IL)‑6 and IL‑1β, and proliferation‑related genes including CyclinD1 and Survivin. The expression of phospho‑p65 was determined by western blotting. Activation of LTβR on bladder cancer 5,637cells was demonstrated to upregulate the mRNA expression levels of the RELA proto‑oncogene, RelA, by 2.5‑fold compared with unstimulated cells, while no significant change was observed in the RELB proto‑oncogene NF‑κB member mRNA levels. Expression of pro‑inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)‑1β mRNA levels were significantly increased nearly 5‑fold and 1.5‑fold, respectively, following LTβR activation compared with unstimulated cells. The LTβR‑induced upregulation of RelA, TNFα and IL‑1β was decreased by ~33, 27, and 26% respectively when LTβR was silenced via short hairpin RNA. Activation of LTβR had no effect on 5,637 cell growth, despite CyclinD1 and Survivin mRNA levels increasing by ~2.7 and 1.3‑fold, respectively, compared with unstimulated cells. In conclusion, activation of LTβR induced the expression of RelA mRNA levels. LTβR activation might be an important mediator in promoting an inflammatory microenvironment in bladder cancer, via the upregulation of TNFα and IL‑1β mRNA levels. LTβR may be a potential therapeutic target for bladder cancer.

Apoptosis-inducing factor (Aif1) mediates anacardic acid-induced apoptosis in Saccharomyces cerevisiae.

Anacardic acid is a medicinal phytochemical that inhibits proliferation of fungal as well as several types of cancer cells. It induces apoptotic cell death in various cell types, but very little is known about the mechanism involved in the process. Here, we used budding yeast Saccharomyces cerevisiae as a model to study the involvement of some key elements of apoptosis in the anacardic acid-induced cell death. Plasma membrane constriction, chromatin condensation, DNA degradation, and externalization of phosphatidylserine (PS) indicated that anacardic acid induces apoptotic cell death in S. cerevisiae. However, the exogenous addition of broad-spectrum caspase inhibitor Z-VAD-FMK or deletion of the yeast caspase Yca1 showed that the anacardic acid-induced cell death is caspase independent. Apoptosis-inducing factor (AIF1) deletion mutant was resistant to the anacardic acid-induced cell death, suggesting a key role of Aif1. Overexpression of Aif1 made cells highly susceptible to anacardic acid, further confirming that Aif1 mediates anacardic acid-induced apoptosis. Interestingly, instead of the increase in the intracellular reactive oxygen species (ROS) normally observed during apoptosis, anacardic acid caused a decrease in the intracellular ROS levels. Quantitative real-time PCR analysis showed downregulation of the BIR1 survivin mRNA expression during the anacardic acid-induced apoptosis.

The mTOR Inhibitor Everolimus Overcomes CXCR4-Mediated Resistance to HDAC Inhibitor Panobinostat through Inhibition of p21 and Mitosis Regulators, Sensitizing MM Cells to DNA-Damaged Induced Apoptosis

Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it.Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis.Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis.Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fish-Oil-Derived DHA-mediated Enhancement of Apoptosis in Acute Lymphoblastic Leukemia Cells is Associated with Accumulation of p53, Downregulation of Survivin, and Caspase-3 Activation

ABSTRACTIn acute lymphoblastic leukemia (ALL), resistance to chemotherapy is associated with inactivation of p53 and upregulation of survivin. Thus, targeting the p53 and survivin expression may provide an attractive strategy for ALL treatment. It has been shown that fish-oil-derived docosahexaenoic acid (DHA) activates several antitumorigenic mechanisms in tumor cells, but little is known regarding the role of DHA on modulating p53 and survivin expression in ALL cells. In this study, we investigated the alterations of the p53 and survivin expression and induction of apoptosis in DHA-treated Molt-4 cells that serve as a model for ALL cells. Molt-4 cells were treated with 50, 100, 150, and 200 μM DHA after which cell proliferation, survivin mRNA and protein levels, p53 protein level, caspase-3 activation, and apoptotic rates were evaluated by different cellular and molecular techniques. After 48- and 72-h treatments with DHA at concentrations ranging from 50 to 200 μM, cell proliferation rates were measured to be 80.5–44.4%, and 73.4–14.4%, respectively, compared to untreated cells. We also found that treatment for 48 h with 200 μM DHA resulted in 10.8- and 3.6-fold increase in p53 protein level and caspase-3 activation followed by 4.7-and 1.6-fold decrease in survivin mRNA and protein levels, respectively, compared to untreated cells. Treatment of cells with different concentrations of DHA dramatically increased the p53/survivin and caspase-3/survivin ratios by 2.8- to 16.9-fold and 3.3 to 5.6-fold increases, respectively, compared to untreated cells. A decrease in the number of cells ranging from 16% to 70% and an increase in the number of apoptotic cells ranging from 9.3% to 93% was also observed with increasing DHA concentrations. In conclusion, p53 and survivin may provide promising targets of DHA in ALL cells and this compound with high proapoptotic capacity represents the possibility of its therapeutic application for ALL treatment.

Expressions and significance of the second mitochondria-derived activator of caspase protein and Survivin in tissues of high-grade intraepithelial neoplasia and esophageal squamous cell carcinoma

Objective To detect the expressions of the second mitochondria-derived activator of caspase protein(Smac) and Survivin in tissues of high-grade intraepithelial neoplasia(HGIN)and esophageal squamous cell carcinoma (ESCC), and explore the influence of Smac and Survivin on occurrence and development of ESCC. Methods The retrospective case-control study was conducted. The clinicopathological data of 45 healthy controls with suspected esophagitis (control group), 41 patients with HGIN (HGIN group) and 57 patients with ESCC (ESCC group) who were admitted to the Qinghai University Affiliated Hospital between January 2012 and December 2014 were collected. Observation indicators included: (1) expressions of Smac and Survivin were detected by immunohistochemistry. (2) Expressions of mRNA of Smac and Survivin in tumor tissues and adjacent tissues were detected by real-time polymerase chain reaction (RT-PCR). (3) Correlation between relative expressions of Smac and Survivin in tumor tissues and adjacent tissues. (4) Relationship between expressions of Smac and Survivin in tumor tissues and clinicopathological factors. Measurement data with normal distribution were represented as ±s. The comparisons among groups was evaluated with the one-way ANOVA. The comparison between tumor tissues and adjacent tissues were analyzed by the paired t test, and comparison of rates was done using the chi-square test and Fisher exact probability. The correlation between expressions of Smac and Survivin was done using the Pearson correlation analysis. Results (1) Expressions of Smac and Survivin were detected by immunohistochemistry. The positive expression rate of Smac of esophageal tissues in the control group, HGIN group and ESCC group were respectively 82.2%(37/45), 43.9%(18/41) and 40.4%(23/57), with a statistically significant difference among the 3 groups (χ2=20.408, P 0.05). The positive expression rate of Smac of adjacent tissues in the ESCC group was 84.2%(48/57), with a statistically significant difference between tumor tissues and adjacent tissues (χ2=20.530, P 0.05). The positive expression rate of Survivin of adjacent tissues in the ESCC group was 15.8%(9/57), with a statistically significant difference between tumor tissues and adjacent tissues (χ2=32.386, P 0.05) and in adjacent tissues (r=-0.197, P>0.05). There was a correlation in the relative expression of Smac between tumor tissues and adjacent tissues (r=0.527, P 0.05). (4) The relationship between the expressions of Smac and Survivin in tumor tissues and clinicopathologic factors: the positive expression of Smac in tumor tissues was 51.6% in men, 9.1% in women, 64.7% in tumor diameter<3 cm, 28.0% in tumor diameter≥3 cm, 60.0% in stage Ⅰ-Ⅱ of TNM stage and 22.7% in stage Ⅲ-Ⅳ of TNM stage, respectively, showing statistically significant differences in the positive expression of Smac in tumor tissues of patients with different gender, tumor diameter and TNM stage (χ2=6.093, 5.567, 6.041, P<0.05). The positive expression of Survivin in tumor tissues was 54.5% in high- and moderate-differentiated tumors, 100.0% in low-differentiated tumors, 75.0% in stageⅠ-Ⅱ and 100.0% in stage Ⅲ-Ⅳ, respectively, showing statistically significant differences in the positive expression of Survivin in tumor tissues of patients with different tumor differentiation and TNM stage (P<0.05). Conclusions The expression of Smac in the progression of esophageal epithelial cells towards to high-grade intraepithelial neoplastia and proliferatoin is inhibited, while expression of Survivin is activated. Smac and Survivin participate in the occurrence of ESCC and maybe play an role in determining the biological features of carcinoma tissues. Key words: Esophageal Neoplasms; Second mitochondria-derived activator of caspase protein; Survivin protein; High-grade esophageal intraepithelial neoplasia

The cannabinoid receptor CB1 contributes to the development of ectopic lesions in a mouse model of endometriosis.

Does signaling via the cannabinoid (CB1) receptor play a role in the pathogenesis of endometriosis in a mouse model? Mice treated with a CB1 agonist developed larger ectopic lesions, while less severe lesions developed in the absence of functional CB1 expression. The expression of components of the endocannabinoid system has been demonstrated in both mouse and human uteri. CB1 receptors are expressed in human epithelial and stromal cell lines derived from eutopic endometrium and deep infiltrating endometriosis nodules. This was a randomized study in a mouse model of endometriosis. In a first set of experiments, mice with endometriosis were treated with the CB1 receptor agonist methanandamide (MET) (5 mg/kg, n = 20) on Days 1-5 and 8-12. In a second set of experiments, endometriosis development was evaluated in CB1-/- mice and in their wild-type (WT) littermates. Endometriosis-like lesions were induced in Balb/c and C57/Bl6 mice. Two weeks after disease induction, the lesions were counted, measured and either included for immunohistochemistry analysis or frozen for gene expression profiling by semi-quantitative real-time PCR. To limit the role of chance, the experiments were conducted under standardized laboratory conditions with appropriate controls. The lesion total volume was significantly higher in MET-treated compared with vehicle-treated mice (P<0.05). Expression levels of mRNA for survivin, N-cadherin, integrin β1 and interleukin-6 were increased in the ectopic endometrium of MET-treated versus vehicle-treated mice (P<0.05). CB1-/- recipients that received endometrial tissue fragments from CB1-/- donors, WT recipients that received endometrial tissue fragments from CB1-/- donors and CB1-/- recipients that received endometrial tissue fragments from WT donors all showed a significant reduction in total lesion volume and lower expression of survivin and N-cadherin compared with WT recipients receiving uterine fragments from WT donors (P<0.05). N/A. We provide evidence that endocannabinoid signaling via CB1 receptor plays a role in the development of endometriosis in a mouse model. However, the relative contribution of the CB1-mediated signaling pathways active in inflammatory, uterine and peritoneal cells remains to be ascertained. Since the study was performed in a mouse model, the significance of the findings in the human system warrants further investigation. Clarifying the function and regulation of CB1 and its molecular interactions with endogenous ligands, and how endocannabinoids levels are regulated in women with endometriosis, represent critical areas of research for the potential development of a novel medical treatment of the disease. A.M.S. was supported by a fellowship from Fondazione Giorgio Pardi. The authors have no conflicts of interest to declare.

Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7.

Nanodiamond (ND) is a renowned material in nonviral small interfering RNA (siRNA) carrier field due to its unique physical, chemical, and biological properties. In our previous work, it was proven that ND could deliver siRNA into cells efficiently and downregulate the expression of desired protein. However, synthesizing a high-efficient tumor-targeting carrier using ND is still a challenge. In this study, a novel carrier, NDCONH(CH2)2NH-VDGR, was synthesized for siRNA delivery, and its properties were characterized with methods including Fourier transform infrared spectrometry, transmission electron microscopy, scanning electron microscopy, gel retardation assay, differential scanning calorimetry, confocal microscopy, releasing test, real-time polymerase chain reaction (PCR) assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, cytotoxicity assay, and gene-silencing efficacy assay in vitro and in vivo. The mechanism of NDCONH(CH2)2NH-VDGR/survivin-siRNA-induced tumor apoptosis was evaluated via flow cytometer assay using Annexin V–fluorescein isothiocyanate/propidium iodide staining method. The NDCONH(CH2)2NH-VDGR/survivin-siRNA nanoparticle with 60–110 nm diameter and 35.65±3.90 mV zeta potential was prepared. For real-time PCR assay, the results showed that the expression of survivin mRNA was reduced to 46.77%±6.3%. The expression of survivin protein was downregulated to 48.49%±2.25%, as evaluated by ELISA assay. MTT assay showed that NDCONH(CH2)2NH-VDGR/survivin-siRNA had an inhibitory effect on MCF-7 cell proliferation. According to these results, the survivin-siRNA could be delivered, transported, and released stably, which benefits in increasing the gene-silencing effect. Therefore, as an siRNA carrier, NDCONH(CH2)2NH-VDGR was suggested to be used in siRNA delivery system and in cancer treatments.

Study of survivin gene expression in hepatocellular carcinoma patients

Objective The aim of this study was to assess the differential expression of survivin gene in tumorous versus nontumorous liver tissue of hepatocellular carcinoma (HCC) patients and determine the possible influence of survivin gene polymorphism −31G/C and +9809T/C on its expression and its association with the risk for HCC in Egyptian patients. Background Mechanisms of hepatic carcinogenesis are not completely understood, but HCC development and progression could be attributed to accumulated genetic alterations as other malignancies. Survivin has functional involvement in apoptosis and proliferation, and is upregulated in malignancy. It attracts considerable interest as a potential diagnostic, prognostic tumor marker and a new target for cancer treatment. Patients and methods This study was performed on 30 HCC patients and 30 unrelated apparently healthy individuals who served as a control group. Genotyping of survivin gene at −31G/C and +9809T/C was performed for HCC cases and controls using TaqMan allelic discrimination Assay. Real-time PCR was performed for survivin mRNA in paired tumorous and nontumorous liver tissue specimens of the HCC patients. Results No significant difference was found between HCC patients and controls as regards different genotypes and alleles of survivin −31G/C and +9809T/C polymorphism. Moreover, none of these genotypes and alleles was associated with risk for HCC. Survivin mRNA was expressed in all tumorous and nontumorous tissue specimens in HCC cases and its expression was significantly higher in tumorous tissues compared with nontumorous tissues by 13 folds. Survivin expression in tumorous tissue was significantly increased with high α-fetoprotein, larger size of the tumor, vascular invasion, absence of cirrhosis, recurrence within 1 year, and low survival rate. The advanced stage of the tumor was the independent variable for survivin expression (P Conclusion Genetic variations of survivin gene had no effect on the susceptibility to HCC in Egyptian patients. Survivin was significantly overexpressed in HCC cases with advanced tumor stage.

Therapeutic Vaccination against A Modified Minimal Survivin Epitope Induces Functional CD4 T Cells That Recognize Survivin‐Expressing Cells

During the last decade, cancer vaccination has received a great deal of attention; however, documentation of the clinical effect remains limited. The introduction of immune checkpoint inhibitors as a novel therapeutic modality 1–4 could by combination increase the ability of a peptide vaccination to induce clinical responses. Survivin is an anti-apoptotic protein, first discovered in 1997. 5 It is overexpressed in a wide array of cancers, including malignant melanoma (MM), 5,6 and expression is correlated with disease progression and a poor prognosis in e.g. colon cancer and neuroblastoma. 7,8 Survivin is an excellent target for cancer vaccines due to its universal expression in cancer; in addition, down-regulation will impair the tumor cell's ability to proliferate and expand. This article is protected by copyright. All rights reserved.

Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells

One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles.Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated.Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs.The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm2), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm2). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation.

Effect of DJ-1 silencing by RNA interference on growth of xenografted human laryngeal squamous cell carcinoma Hep-2 cells in nude mice

Objective: To investigate the effect of silencing DJ-1 on xenografted human laryngeal squamous cell carcinoma (LSCC) Hep-2 cells in nude mice. Methods: Xenograft model of human LSCC was established by subcutaneous transplantation of Hep-2 cells in 24 nude mice. The LSCC-bearing nude mice were randomly divided into 3 groups (n=8 in each):DJ-1 siRNA low dose group and DJ-1 siRNA high dose group were injected in tumors with 20 μg of DJ-1 siRNA or 40 μg of DJ-1 siRNA in 50 μL, respectively; control group was injected with 5% glucose solution in 50 μL, twice a week for 3 weeks. The weight and size of tumors were measured before injection. The animals were sacrificed 48 h after the final treatment, and the tumors were harvested and weighed. The apoptosis and proliferation of tumor cells were determined; the expressions of Caspase-3 and Ki-67 in tumor specimens were detected with immunohistochemistry. The expression of DJ-1, PTEN, survivin mRNA and protein in tumor tissues were detected by RT-PCR and Western blotting, respectively. Results: Tumor weight in low dose group[(0.66±0.15)g] and high dose group[(0.48±0.11)g] were significantly lower than that in control group[(0.83±0.16)g, all P<0.05]. The inhibition rates of low dose group and high dose group were (20.48±0.18)% and (42.16±0.13)%, respectively. Immunohistochemistry showed that the expression of Caspase-3 was increased and Ki-67 was reduced in tumor specimens, compared with the control group (all P<0.05). RT-PCR and Western blot results showed that in low dose group and high dose group the mRNA and protein expression of DJ-1 and survivin significantly decreased (all P<0.05), while PTEN mRNA and protein content increased (all P<0.05). Conclusion: High dose DJ-1 siRNA can inhibit the tumor growth in human LSCC xenograft nude mouse model, which indicates that down-regulating DJ-1 and survivin, and up-regulating PTEN expression may lead to blockage of PI3K-PKB/Akt signaling pathway and promoting tumor cell apoptosis.

DNA Strand Replacement Mechanism in Molecular Beacons Encoded for the Detection of Cancer Biomarkers.

Signaling properties of a fluorescent hairpin oligonucleotide molecular beacon (MB) encoded to recognize protein survivin (Sur) mRNA have been investigated. The process of complementary target binding to SurMB with 20-mer loop sequence is spontaneous, as expected, and characterized by a high affinity constant (K = 2.51 × 10(16) M(-1)). However, the slow kinetics at room temperature makes it highly irreversible. To understand the intricacies of target binding to MB, a detailed kinetic study has been performed to determine the rate constants and activation energy Ea for the reaction at physiological temperature (37 °C). Special attention has been paid to assess the value of Ea in view of reports of negative activation enthalpy for some nucleic acid reactions that would make the target binding even slower at increasing temperatures in a non-Arrhenius process. The target-binding rate constant determined is k = 3.99 × 10(3) M(-1) s(-1) at 37 °C with Ea = 28.7 ± 2.3 kcal/mol (120.2 ± 9.6 kJ/mol) for the temperature range of 23 to 55 °C. The positive high value of Ea is consistent with a kinetically controlled classical Arrhenius process. We hypothesize that the likely contribution to the activation energy barrier comes from the SurMB stem melting (tm = 53.7 ± 0.2 °C), which is a necessary step in the completion of target strand hybridization with the SurMB loop. A low limit of detection (LOD = 2 nM) for target tDNA has been achieved. Small effects of conformational polymorphs of SurMB have been observed on melting curves. Although these polymorphs could potentially cause a negative Ea, their effect on kinetic transients for target binding is negligible. No toehold preceding steps in the mechanism of target binding were identified.