Survivin mRNA Expression Research Articles

The effect of Yiqi-jianpi-jiedu recipe decoction containing serum on oxaliplatin-induced HepG2 human hepatoma cell

Objective To explore the effect of Yiqi-jianpi-jiedu recipe decoction containing serum on Oxaliplatin-induced HepG2 Human hepatoma cell survivin, caspase-3, caspase-9 expression. Methods The serum pharmacological method was used to prepare nourishing Qi and invigorating spleen decoction containing serum of rat. The HepG2 human hepatoma cells were divided into Chinese herb containing serum group, chemotherapy with Oxaliplatin (OXA) group and the combination group (medicine containing serum and OXA), and the blank group without drugs. Apply MTT assay, Western Blotting method and RT-PCR method to detect the cell inhibition rate and survivin, caspase-3, caspase-9 protein and gene expression changes. Results Compared with the blank group, the inhibition rate of HepG2 Human hepatoma cell was significantly higher in Chinese herb containing serum group, chemotherapy group and the combination group (P < 0.05). Compared with thechemotherapy group and Chinese herb containing serum group, the Survivin (0.151 ± 0.054 vs. 0.288 ± 0.089, 0.375 ± 0.063) and the expression of Survivin mRNA (0.205 ± 0.091 vs. 0.487 ± 0.073, 0.725 ± 0.092) of the conbination group downgraded significantly lower (P < 0.05 or P< 0.01). Compared with chemotherapy group and Chinese herb containing serum group, the caspase-3 (0.821 ± 0.079 vs. 0.634 ± 0.098, 0.487 ± 0.102), caspase-9 (0.901 ± 0.047 vs. 0.709 ± 0.054, 0.402 ± 0.012) expression, caspase-3 mRNA (1.928 ± 0.226 vs. 1.564 ± 0.195, 1.287 ± 0.312) and caspase-9 mRNA (2.063 ± 0.517 vs. 1.536 ± 0.084, 1.019 ± 0.182) expression of the conbination group upgraded significantly higher (P < 0.05 or P < 0.01). Conclusions Nourishing-Qi and invigorating spleen decoction containing serum may affect Oxaliplatin-induced HepG2 apoptosis mechanism through increasing the suppression on Survivin and promoting the expression of caspase-3 and caspase-9 protein and genes. This research provided important experimental basis for clinical application of traditional Chinese medicine treating liver cancer. Key words: Apoptosis; Seropharmacology (TCD); Yiqi-jianpi-jiedu recipe

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.

Effects of leptin on proliferation and apoptosis of colorectal carcinoma SW480 cells

Objective To observe the effects of leptin on proliferation and apoptosis of colorectal carcinoma SW480 cells, and to explore its possible mechanism. Methods After stimulation with various doses of leptin (0, 20, 50, 100, 200 ng/ml) for different durations (12, 24, 36 and 48 h), the cell proliferation rate was determined by cell counting kit-8 (CCK-8) assay. Serum deprivation was used to induce apoptosis of SW480 cells stimulated with leptin. Cell apoptosis was assessed by double staining with Annexin V and flow cytometry. The mRNA expression of Survivin and beta-catenin in SW480 cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Results As compared with the control group, with the increase of leptin concentration (20, 50, 100 and 200 ng/ml), the proliferation rate of colorectal cancer SW480 cells [12 h: (118.1±2.0)%, (132.6±3.9)%, (141.9±1.3)% and (145.6±4.9)%, P=0.000, 0.000, 0.000, 0.000; 24 h: (120.8±2.7)%, (137.0±1.0)%, (149.7±1.6)% and (176.6±2.9)%, P=0.000, 0.000, 0.000, 0.000; 36 h: (123.9±0.5)%, (133.9±0.8)%, (169.4±1.1)% and (182.8±1.1)%, P=0.001, 0.001, 0.000, 0.000; 48 h: (132.7±1.2)%, (146.0±3.3)%, (172.0±0.6)% and (185.5±0.5)%, P=0.001, 0.005, 0.000, 0.000] was increased (P<0.05), and the serum starvation-induced apoptosis rate [(30.23±0.15)%, (28.87±1.01)%, (18.23±0.35)%, (16.33±0.51)% and (9.20±0.26)%] was decreased (P=0.388, 0.000, 0.001, 0.000). The mRNA expression levels of Survivin and beta-catenin were increased (P=0.007, 0.003, 0.000, 0.000; P=0.000, 0.000, 0.000, 0.000). Conclusion Leptin could promote the proliferation and inhibit the apoptosis of SW480 cells. Key words: Leptin; Colorectal carcinoma; Proliferation; Apoptosis

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.

Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth.

Transcription factor Specificity protein1 (Sp1) and its downstream target survivin (inhibitor of apoptosisprotein), play major roles in the pathogenesis of various cancers. Ewing Sarcoma (ES) is a common soft tissue/bone tumor in adolescent and young adults. Overexpression of survivin is also linked to the aggressiveness and poor prognosis of ES. Small molecule Tolfenamic acid (TA) inhibits Sp1 and survivin in cancer cells. In this investigation, we demonstrate a strategy to target Sp1 and survivin using TA and positive control Mithramycin A (Mit) to inhibit ES cell growth. Knock down of Sp1 using small interfering RNA (siRNA) resulted in significant (p<0.05) inhibition of CHLA-9 and TC-32 cell growth as assessed by CellTiter-Glo assay kit. TA or Mit treatment caused dose/time-dependent inhibition of cell viability, and this inhibition was correlated with a decrease in Sp1 and survivin protein levels in ES cells. Quantitative PCR results showed that Mit treatment decreased the mRNA expression of both survivin and Sp1, whereas TA diminished only survivin but not Sp1. Proteasome inhibitor restored TA-induced inhibition of Sp1 protein expression suggesting that TA might cause proteasome-dependent degradation. Gel shift assay using ES cell nuclear extract and biotinylated Sp1 consensus oligonucleotides confirmed that both TA and Mit decreased DNA-binding activity of Sp1. These results demonstrate that both Mit and TA reduce expression of Sp1 and survivin, disrupt Sp1 DNA-binding and inhibit ES cell proliferation. This investigation suggests that targeting Sp1 and survivin could be an effective strategy for inhibiting ES cell growth.

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.

The cytotoxic effect of GW843682X on nasopharyngeal carcinoma.

GW843682X is a publicly available anti-cancer compound by inhibiting Polo-like kinase. Previous studies revealed that GW843682X inhibited the proliferation of various tumor cell lines. In this study, the cytotoxic effect of GW843682X was investigated on cell proliferation, cell cycle and apoptosis of nasopharyngeal carcinoma 5-8F cells. Cell morphological changes were observed by inverted microscopy. Cell proliferation was tested by CCK8 assay. Cell cycle arrest and apoptosis were tested by flow cytometry. The mechanism of apoptosis was investigated by RT-PCR to determine the mRNA expression of IAP-1, IAP-2, XIAP, and survivin. GW843682X resulted in remarkable cell morphological changes with the increase of drug concentrations. CCK8 assay revealed that GW843682X inhibited the proliferation and induced apoptosis of 5-8F cells in a dose-dependent manner (IC50=62.5-125nmol/L). After treating 5-8F cells with different doses of GW843682X for 12 h, G2/M phase cells significantly increased while G0/G1 phase cells remarkably decreased. Interestingly, GW843682X significantly inhibited the mRNA expression of IAP-1 and survivin, which function as key regulators of mitosis and programmed cell death, and is overexpressed in many tumor types. The mechanism of cytotoxic effect is partially due to the inhibition of IAP gene expression. These findings indicated that GW843682X exhibited remarkable cytotoxic effects on nasopharyngeal carcinoma 5-8F cells by down-regulating IAP gene expression, suggesting that GW843682X may become a novel therapeutic agent for nasopharyngeal carcinoma.

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.

A Study of Gene Expression of Survivin, its Antiapoptotic Variants, and Targeting Survivin In Vitro for Therapy in Retinoblastoma.

Apoptosis is a natural process regulated by apoptotic and antiapoptotic molecules. We investigated mRNA expression of survivin and its splice variants, along with B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax), in a cohort of 20 retinoblastoma (RB) tumors by real-time polymerase chain reaction. We hypothesized a correlation between the Bcl-2/Bax and survivin splice variants and also that expression of these would be associated with clinicopathologic features of tumors. The Bcl-2 expression was significantly higher (P<0.001) in RB, and Bcl-2/Bax ratio was remarkably higher in poorly differentiated tumors. A statistically significant higher expression of Survivin-WT (wild type) compared with its variant Survivin-2β (P<0.05) was observed. Bcl-2 did not exhibit positive correlation with any of the survivin variants except Survivin-2β, whereas Bax exhibited significant (P<0.05) correlation with the variants. Thus, it could be suggested that a superior player out of a likely interaction between the variants and Bcl-2/Bax uses its activity for the progression of RB. Silencing of Survivin-WT in the Y79 cell line was studied by siRNA technology and cell-permeable dominant negative survivin (SurR9-C84A). siRNA showed higher proapoptotic effects and increased caspase 3/7 activity in Y79 cells. Effective internalization of SurR9-C84A in Y79 cells induced cytotoxic effects. Thus, the current study confirms survivin as a promising target for therapy.

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.

Arsenic Trioxide Induces Apoptosis and Incapacitates Proliferation and Invasive Properties of U87MG Glioblastoma Cells through a Possible NF-κB-Mediated Mechanism.

Identification of novel therapeutics in glioblastoma remains crucial due to the devastating and infiltrative capacity of this malignancy. The current study was aimed to appraise effect of arsenic trioxide (ATO) in U87MG cells. The results demonstrated that ATO induced apoptosis and impeded proliferation of U87MG cells in a dosedependent manner and also inhibited classical NF-κB signaling pathway. ATO further upregulated expression of Bax as an important proapoptotic target of NF-κB and also inhibited mRNA expression of survivin, c-Myc and hTERT and suppressed telomerase activity. Moreover, ATO significantly increased adhesion of U87MG cells and also diminished transcription of NF-κB down-stream targets involved in cell migration and invasion, including cathepsin B, uPA, MMP-2, MMP-9 and MMP-14 and suppressed proteolytic activity of cathepsin B, MMP-2 and MMP-9, demonstrating a possible mechanism of ATO effect on a well-known signaling in glioblastoma dissemination. Taken together, here we suggest that ATO inhibits survival and invasion of U87MG cells possibly through NF-κB-mediated inhibition of survivin and telomerase activity and NF-κB-dependent suppression of cathepsin B, MMP-2 and MMP-9.

Highly stable polyglutamate derivatives/siRNA polyplex efficiently down-relegate survivin expression and augment the efficacy of cisplatin

RNA interfere (RNAi)-based technology holds great promise in cancer treatment. The use of small interfering RNA (siRNA), however, is hampered by its low delivery efficiency in vivo when they are diluted in blood biofluids and in the presence of serum and salt. In this study, we developed the polyglutamate derivative polymer brush, poly(ethyleneglycol) monomethyl ether-b-polyglutamate-g-spermine (mPEG-b-PG-g-spermine, PPGS), which could efficiently deliver survivin-siRNA under ultra-high dilution and in the presence of salt (NaCl 150mM) and serum (10% FBS), most likely due to its PEG-shelled polymer brush structure. On the contrary, aggregation occurred when PEI/siRNA polyplex dispersed in saline and serum-containing media and PEI polyplex dissociated after making a 256-fold dilution. PPGS/si-survivin polyplex exhibited high cellular uptake efficiency and efficiently down-regulated the expression of survivin mRNA in the cisplatin-resistance of non-small cell human lung adenocarcinoma (A549/DDP) cells in the presence of serum. However, either PEI polyplex or Lipofectmine 2000 complex was unstable in serum and salt-containing media and at high dilution rates, which resulted in their dramatical decrease of cellular uptake and gene-silencing efficiency in these conditions. The PPGS/si-survivin polyplex also exhibited synergistic effects of killing the cancer cells by combination treatment with cisplatin. Therefore, the PPGS gene carrier showed great potential in systemic siRNA delivery, and its combination with chemotherapeutic drug is promising in treating drug resistant cancers.

Effect and mechanism of miR-34a on proliferation, apoptosis and invasion of laryngeal carcinoma cells.

To discuss the effect and mechanism of miR-34a on the proliferation, apoptosis and invasion of laryngeal carcinoma cells. The laryngeal squamous carcinoma Hep2 cells were transiently transfected with miR-34a mimics and miR-34a NC. The MTT, colony-forming assay, Hoechst staining and AnnexinV-PI double staining flow cytometry were employed to detect the effect of miR-34a on the viability and apoptosis of laryngeal squamous carcinoma Hep2 cells; Transwell assay to defect the effect of miR-34a on the migration and invasion of laryngeal squamous carcinoma Hep2 cells; western blot and RT-PCR assay to defect the effect of miR-34a mimics on the expression of survivin and Ki-67 mRNA in laryngeal squamous carcinoma Hep2 cells. Compared with miR-34a NC group, the cell viability in miR-34 mimics group was significantly decreased (P<0.01), the cell apoptosis rate was significantly increased (P<0.01), the abilities of cell migration and invasion were significantly reduced (P<0.01) and the expression of survivin and Ki-67 mRNA was significantly decreased (P<0.01). The increased expression of miR-34a can induce the apoptosis of Hep2 laryngeal carcinoma cells and inhibit the cell proliferation and invasion, which is related to the down-regulated expression of survivin and Ki-67.

The anti-cancer effect of quercetin in renal cancer through regulating survivin expression and caspase 3 activity

Background: Traditional chemotherapy and radiotherapy are ineffective in the treatment of renal cancer. Quercetin exhibits a broad-,spectrum anti-cancer role with only minor side effects in the treatment of many tumors, but its efficacy in renal cancer treatment is presently unknown.Methods: Renal cancer ACHN cells were cultured and treated with quecertin at various dosages. Cell viability was tested by MTT assay. Flow cytometry measured cell apoptosis. Real time PCR and Western Blot measured survivin expression, and caspase 3 activity was also detected.Results: Quecertin significantly suppressed ACHN cell proliferation and induced early cell apoptosis in a dose-dependent manner (p < 0.05). Quecertin significantly enhanced caspase 3 activity (p < 0.05), but downregulated survivin mRNA and protein expression (p < 0.05).

Differential effects of a high-fat diet on serum lipid parameters and ovarian gene expression in young and aged female mice.

The aim of this study was to compare serum lipid profiles and ovarian gene expression between aged and younger female mice fed a control or a high-fat diet for 2 months. For this 16 female mice (C57BL/6) of 4 months (Young, n = 8) or 13 months (Old, n = 8) of age were used. The females were divided into four groups: (i) young females fed a normal diet; (ii) young females fed a high-fat diet; (iii) old females fed a normal diet; and (iv) old females fed a high-fat diet. Food intake was reduced (P < 0.05) in mice fed with a high-fat (2.9 ± 0.1 g) diet in comparison with control mice (3.9 ± 0.1 g). Body weight was higher for old females on the high-fat diet (35.1 ± 0.3 g) than for young females on the same diet (23.3 ± 0.4 g; P < 0.05). PON1 activity was lower in the high-fat than control diet group (114.3 ± 5.8 vs. 78.1 ± 6.0 kU/L, respectively) and was higher in older than younger females (85.9 ± 6.4 vs. 106.5 ± 5.3; P < 0.05, respectively). Females fed a high-fat diet had lower expression of Igf1 mRNA (P = 0.04). There was an interaction between age and diet for the expression of Gdf9 and Survivin, with lower expression in older females in both diets and young females that received the high-fat diet (P < 0.05). Concluding, the high-fat diet reduced the expression of ovarian Igf1 mRNA, and Gdf9 and Survivin mRNA in younger females, which can indicate lower fertility rates. High-density lipoprotein concentration and PON1 activity were higher in aged female mice.

MiR-23b targets cyclin G1 and suppresses ovarian cancer tumorigenesis and progression.

BackgroundIt has been proposed that cyclin G1 (CCNG1) participates in p53-dependent G1–S and G2 checkpoints and might function as an oncogenic protein in the initiation and metastasis of ovarian carcinoma. MicroRNA 23b (miR-23b) is a critical regulatory factor in the progression of many cancer cell types that targets the relevant genes.MethodsMiR-23b expression in ovarian tissues was quantified by quantitative reverse transcription–PCR. The ovarian cancer cell lines OVCAR3, HO8910-PM, and SKOV3/DDP were transfected with miR-23b, after we assayed the cell phenotype and expression of the relevant molecules. Dual-luciferase reporter assay and a xenograft mouse model were used to examine the expression of miR-23b and its target gene CCNG1.ResultsMIR23B mRNA expression was significantly lower in epithelial ovarian carcinoma and borderline tumors than in normal ovarian tissues and benign tumors, and miR-23b expression among ages and pathological subtypes was significantly different. CCNG1 mRNA expression was significantly lower in normal ovarian tissues than in benign tumors, borderline tumors, and ovarian carcinomas, and expression among pathological subtypes was significantly different. MiR-23b overexpression inhibited ovarian cancer cell proliferation, invasion, and migration, and induced apoptosis. Dual-luciferase reporter assay showed that miR-23b bound with the 3′ untranslated region of CCNG1. MiR-23b overexpression significantly downregulated CCNG1, urokinase, survivin, Bcl-xL, P70S6K, and matrix metallopeptidase-9 (MMP9) mRNA and protein expression. Furthermore, miR-23b inhibited tumor growth and suppressed CCNG1 expression in vitro.ConclusionsOur findings show that miR-23b may inhibit ovarian cancer tumorigenesis and progression by downregulating CCNG1 and the expression of the relevant genes. MiR-23b is a potentially novel application for regulating ovarian carcinoma progression.

Resveratrol Enhances Apoptosis in Endometriotic Stromal Cells.

Resistance to apoptosis, together with inflammatory and invasive activity, contributes to the pathogenesis of endometriosis; therefore, approaches that can safely enhance apoptosis in endometriotic tissue are highly sought after as a means of managing the disease. Although resveratrol (RVT) is known to induce apoptosis or increase sensitivity to apoptotic stimuli in various cancer cell types, its effect on human endometriosis has remained uncertain. This study aimed to investigate whether RVT induces or enhances apoptosis in human endometriotic stromal cells (ESCs). Endometriotic tissues were collected, during laparoscopies, from women affected by ovarian endometriosis. ESCs were prepared, cultured, and treated with RVT. Apoptosis was assessed by annexin V-PI staining. Survivin mRNA expression in ESCs was examined using RT-PCR. ESCs were pre-treated with or without RVT and then incubated with TNF-α-related-apoptosis-inducing ligand (TRAIL), which is a known pro-apoptotic molecule. RVT alone did not induce apoptosis in ESCs. RVT significantly reduced survivin mRNA expression (P < 0.05). Pre-treatment with RVT significantly enhanced TRAIL-induced apoptosis (8.13 ± 0.83% (control) versus 29.19 ± 7.39% (pre-treated with RVT), P < 0.05). This study indicates that RVT suppresses survivin expression and enhances TRAIL-induced apoptosis in ESCs.

Ochratoxin A mediates MAPK activation, modulates IL-2 and TNF-α mRNA expression and induces apoptosis by mitochondria-dependent and mitochondria-independent pathways in human H9 T cells.

Ochratoxin A (OTA) is a natural fungal secondary metabolite that contaminates food and animal feed. Human exposure and involvement of this mycotoxin in several pathologies have been demonstrated worldwide. We investigated OTA immunotoxicity on H9 cells, a human cutaneous CD4+ T lymphoma cell line. Cells were treated with 0, 1, 5, 10, and 20 µM OTA for up to 24 hr. Western blotting revealed increased phosphorylation of all three major mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun amino-terminal kinase, p38). OTA triggered mitochondrial transmembrane potential loss and caspase-3 activation. The 24-hr OTA treatment caused marked changes in cell morphology and DNA fragmentation, suggesting the occurrence of apoptotic events that involved a mitochondria-dependent pathway. Moreover, OTA triggered significant modulation of survivin, interleukin 2 (IL-2) and tumor necrosis factor α (TNF-α): mRNA expression of survivin and IL-2 were decreased, while TNF-α was increased. OTA also caused caspase-8 activation in a time-dependent manner, which evokes the death receptor pathway activation; we suspect that this occurred via the autocrine pro-apoptotic effect of TNF-α on H9 cells.