Survivin mRNA Levels Research Articles

Synthesis and Validation of TRIFAPYs as a Family of Transfection Agents for Therapeutic Oligonucleotides.

Transfection agents play a crucial role in facilitating the uptake of nucleic acids into eukaryotic cells offering potential therapeutic solutions for genetic disorders. However, progress in this field needs the development of improved systems that guarantee efficient transfection. Here, we describe the synthesis of a set of chemical delivery agents (TRIFAPYs) containing alkyl chains of different lengths based on the 1,3,5-tris[(4-alkyloxy-1pyridinio)methyl]benzene tribromide structure. Their delivery properties for therapeutic oligonucleotides were evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs) as a silencing tool. The binding of liposomes to PPRHs was evaluated by retardation assays in agarose gels. The complexes had a size of 125 nm as determined by DLS, forming well-defined concentrical vesicles as visualized by Cryo-TEM. The prostate cancer cell line PC-3 was used to study the internalization of the nanoparticles by fluorescence microscopy and flow cytometry. The mechanism of entrance involved in the cellular uptake was mainly by clathrin-mediated endocytosis. Cytotoxicity analyses determined the intrinsic toxicity caused by each TRIFAPY and the effect on cell viability upon transfection of a specific PPRH (HpsPr-C) directed against the antiapoptotic target survivin. TRIFAPYs C12-C18 were selected to expand these studies in the breast cancer cell line SKBR-3 opening the usage of TRIFAPYs for both sexes and, in the hCMEC/D3 cell line, as a model for the blood-brain barrier. The mRNA levels of survivin decreased, while apoptosis levels increased upon the transfection of HpsPr-C with these TRIFAPYs in PC-3 cells. Therefore, TRIFAPYs can be considered novel lipid-based vehicles for the delivery of therapeutic oligonucleotides.

Lead exposure induces neuronal apoptosis via NFκB p65/RBBP4/Survivin signaling pathway

Lead (Pb), as a heavy metal that is easily exposed in daily life, can cause damage to various systems of body. Apoptosis is an autonomous cell death process regulated by genes in order to maintain the stability of internal environment, which plays an important role in the development of nervous system. RB binding protein 4 (RBBP4) is one of the core histone binding subunits and is closely related to the apoptosis process of nervous system cells. However, it is not known whether RBBP4 can regulate neuronal apoptosis in lead-exposed environments. We exposed PC12 cells to 0 μM (control group), 1 μM, and 100 μM PbAc for 24 h to obtain cell samples. The female rats ingested drinking water containing 0, 0.5 g/L, and 2.0 g/L PbAc from the first day of pregnancy to three weeks after delivery to obtain hippocampal tissue samples from mammary rats. The results of TUNEL showed that lead exposure promoted the onset of apoptosis in cells and hippocampus. The mRNA and protein levels of the apoptosis-related protein Survivin were significantly reduced in the lead-exposed group compared to the control group. In addition, we found that lead exposure reduces the mRNA and protein levels of RBBP4 in PC12 cells and hippocampus, and increases the mRNA and protein levels of NFκB p65. Moreover, inhibiting NFκB p65 can reverse the decrease in RBBP4 expression in the lead exposure model. Overexpression of RBBP4 increased Survivin expression and reduced apoptosis induced by lead exposure. This suggests that lead exposure induces apoptosis through the NFκB p65/RBBP4/Survivin signaling pathway.

Inhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis.

Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/β-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear β-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.

Isoalantolactone mediates the degradation of BCR-ABL protein in imatinib-resistant CML cells by down-regulating survivin

ABSTRACT Isoalantolactone (Iso) is a bioactive lactone isolated from the root of Inula helenium L, which has been reported to have many pharmacological effects. To investigate the role and mechanism of isoalantolactone in chronic myeloid leukemia (CML), we first investigated isoalantolactone’s anti-proliferative effects on imatinib-sensitive and imatinib-resistant CML cells by CCK8. Flow cytometry was used to detect isoalantolactone-induced cell apoptosis. Survivin was overexpressed in KBM5 and KBM5T315I cells using the lentivirus vector pSIN-3×flag-PURO. In KBM5 and KBM5T315I cells, shRNA was used to knockdown survivin. Cellular Thermal Shift Assay (CETSA) was used to detect the interaction between isoalantolactone and survivin. The ubiquitin of survivin induced by isoalantolactone was detected through immunoprecipitation. Quantitative polymerase-chain reaction (Q-PCR) and western blotting were used to detect the levels of mRNA and protein. Isoalantolactone inhibits the proliferation and promotes apoptosis of imatinib-resistant CML cells. Although isoalantolactone inhibits the proteins of BCR-ABL and survivin, it cannot inhibit survivin and BCR-ABL mRNA levels. Simultaneously, it was shown that isoalantolactone can degrade survivin protein by increasing ubiquitination. It was demonstrated that isoalantolactone-induced survivin mediated downregulation of BCR-ABL protein. It was also revealed that isoalantolactone triggered BCR-ABL protein degradation via caspase-3. Altogether, isoalantolactone inhibits survivin through the ubiquitin proteasome pathway, and mediates BCR-ABL downregulation in a caspase-3 dependent manner. These data suggest that isoalantolactone is a natural compound, which can be used as a potential drug to treat TKI-resistant CML.

Significance of Nuclear Factor-Kappa B (NF-κB) and Survivin in Breast Cancer and Their Association with Radiosensitivity and Prognosis.

Analyze the expression of NF-κB and survivin genes and mRNAs in breast cancer, and evaluate their impact on prognosis. Investigate their association with radiosensitivity in breast cancer. The expression levels of NF-κB and survivin genes in breast cancer were analyzed by bioinformatics, NF-κB and survivin mRNA was verified by RTRCR, and their association with prognosis were assessed. Knockdown of survivin by siRNA was used to analyze its association with radiosensitivity in breast cancer. The gene expression of NFKB1 and BIRC5 are differentially expressed in a variety of tumours and their corresponding normal tissue species. In breast cancer tissues, NFKB1 expression levels were reduced compared to normal tissue, while BIRC5 expression levels were increased (P<0.05). In different molecular subtypes of breast cancer, NFKB1 and BIRC5 were differentially expressed (P<0.05), NFKB1 was highly expressed in the luminal subtype and BIRC5 was highly expressed in the TNBC subtype. In TNBC subtype, NFKB1 expression is higher in IM subtype than other subtypes (P<0.05), and BIRC5 expression is higher in BL-2 than other subtypes (P<0.05). NFKB1 was not associated with tumour size, lymph node stage and distant metastasis (P≥0.05), while BRIC5 was associated with these clinical features (P<0.05). NF-κB and survivin genes were negatively correlated (R = - 0.193, P<0.05). The mRNA levels of NF-κB and survivin are expressed in the same trend in breast cancer patients. NF-κB and survivin were not significantly different in recurrent and non-recurrent patients (P≥0.05). The mRNA levels of the both were not correlated with breast cancer subtypes (P≥0.05). The mRNA expression of NF-κB and survivin correlated with distant metastasis. NF-κB and survivin mRNAs were positively correlated (R=0.903, P<0.05). Gene and mRNA expression of NF-κB and survivin were not associated with patients' survival overall survival (OS) (P≥0.05). Down-regulation of survivin has little effect on the proliferation rate of breast cancer cells (P≥0.05), but increase the apoptosis rate of breast cancer cells (P<0.05).The proliferation rate of cells decreased and the apoptosis rate increased significantly (P<0.05) after the implementation of radiotherapy, and this technique could improve the radiosensitivity of breast cancer cells. NF-κB and survivin interact at the gene and mRNA levels. Regulation of mRNA expression of NF-κB or survivin may help to improve the radiosensitivity of breast cancer cells, more experiments are needed to verify this in the future.

Trioleyl Pyridinium, a Cationic Transfection Agent for the Lipofection of Therapeutic Oligonucleotides into Mammalian Cells

One of the most significant limitations that therapeutic oligonucleotides present is the development of specific and efficient delivery vectors for the internalization of nucleic acids into cells. Therefore, there is a need for the development of new transfection agents that ensure a proper and efficient delivery into mammalian cells. We describe the synthesis of 1,3,5-tris[(4-oelyl-1-pyridinio)methyl]benzene tribromide (TROPY) and proceeded to the validation of its binding capacity toward oligonucleotides, the internalization of DNA into the cells, the effect on cell viability, apoptosis, and its capability to transfect plasmid DNA. The synthesis and chemical characterization of TROPY, which can bind DNA and transfect oligonucleotides into mammalian cells through clathrin and caveolin-mediated endocytosis, are described. Using a PPRH against the antiapoptotic survivin gene as a model, we validated that the complex TROPY-PPRH decreased cell viability in human cancer cells, increased apoptosis, and reduced survivin mRNA and protein levels. TROPY was also able to stably transfect plasmid DNA, as demonstrated by the formation of viable colonies upon the transfection of a dhfr minigene into dhfr-negative cells and the subsequent metabolic selection. TROPY is an efficient transfecting agent that allows the delivery of therapeutic oligonucleotides, such as PPRHs and plasmid DNA, inside mammalian cells.

PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

Protein diversity due to alternative mRNA splicing or post-translational modifications (PTMs) plays a vital role in various cellular functions. The mitotic kinases polo-like kinase 1 (PLK1) and Aurora B (AURKB) phosphorylate survivin, an inhibitor of apoptosis (IAP) family member, thereby regulating cell proliferation. PLK1, AURKB, and survivin are overexpressed in triple-negative breast cancer (TNBC), an aggressive breast cancer subtype. TNBC is associated with high proliferative capacity, high rates of distant metastasis, and treatment resistance. The proliferation-promoting protein survivin and its activating kinases, PLK1 and AURKB, are overexpressed in TNBC. In this study, we investigated the role of survivin phosphorylation in racial disparities in TNBC cell proliferation. Analysis of TCGA TNBC data revealed higher expression levels of PLK1 (P = 0.026) and AURKB (P = 0.045) in African Americans (AAs; n = 41) than in European Americans (EAs; n = 86). In contrast, no significant racial differences in survivin mRNA or protein levels were observed. AA TNBC cells exhibited higher p-survivin levels than EA TNBC cells. Survivin silencing using small interfering RNAs significantly attenuated cell proliferation and cell cycle progression in AA TNBC cells, but not in EA TNBC cells. In addition, PLK1 and AURKB inhibition with volasertib and barasertib significantly inhibited the growth of AA TNBC xenografts, but not of EA TNBC tumors. These data suggest that inhibition of PLK1 and AURKB suppresses cell proliferation and tumor growth, specifically in AA TNBC. These findings suggest that targeting survivin phosphorylation may be a viable therapeutic option for AA patients with TNBC.

Anti-tumor effects of Auraptene through induction of apoptosis and oxidative stress in a mouse model of colorectal cancer

The main strategy of cancer cells for survival is uncontrolled cell division and escape from apoptosis. The use of anticancer agents inducing the production of reactive oxygen species (ROS) and controlling cell division might be a therapeutic approach to eradicate cancer cells. Herein, we examined the therapeutic effects of Auraptene on CT26 cells as well as on a mouse model of colorectal cancer (CRC). The spheroid assay was also conducted to analyze the anti-proliferative activity of Auraptene. We also assessed the in vitro analysis of ROS generation. The impact of Auraptene on oxidant/antioxidant markers, as well as the mRNA expression of Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin genes, was evaluated by qPCR in tumor samples. As a result, Auraptene significantly reduced the size of CT26 spheroids at a dose of 200 µM. After 12 h, ROS levels were significantly elevated in CT26 cells. The administration of Auraptene induced apoptosis and the cell cycle arrest by modulating Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin mRNA levels. Furthermore, our results demonstrated that Auraptene suppressed CAT, GSH (reduced Glutathione), and FRAP while increasing MDA in tissue homogenates which in turn could raise oxidative stress and stimulate apoptosis. Therefore, Auraptene may act as a powerful adjuvant therapy in CRC since it triggers apoptosis and cell cycle.

Significance of the Wnt canonical pathway in radiotoxicity via oxidative stress of electron beam radiation and its molecular control in mice

Purpose Radiation triggers cell death events through signaling proteins, but the combined mechanism of these events is unexplored The Wnt canonical pathway, on the other hand, is essential for cell regeneration and cell fate determination. Aim The relationship between the Wnt pathway's response to radiation and its role in radiotoxicity is overlooked, even though it is a critical molecular control of the cell. The Wnt pathway has been predicted to have radioprotective properties in some reports, but the overall mechanism is unknown. We intend to investigate how this combined cascade works throughout the radiation process and its significance over radiotoxicity. Materials and methods Thirty adult mice were irradiated with electron beam radiation, and 5 served as controls. Mice were sacrificed after 24 h and 30 days of irradiation. We assessed DNA damage studies, oxidative stress parameters, mRNA profiles, protein level (liver, kidney, spleen, and germ cells), sperm viability, and motility. Observation The mRNA profile helps to understand how the combined cascade of the Wnt pathway and NHEJ work together during radiation to combat oxidative response and cell survival. The quantitative examination of mRNA uncovers unique critical changes in all mRNA levels in all cases, particularly in germ cells. Recuperation was likewise seen in post-30 day’s radiation in the liver, spleen, and kidney followed by oxidative stress parameters, however not in germ cells. It proposes that reproductive physiology is exceptionally sensitive to radiation, even at the molecular level. It also suggests the suppression of Lef1/Axin2 could be the main reason for the permanent failure of the sperm function process. Post-irradiation likewise influences the morphology of sperm. The decrease in mRNA levels of Lef1, Axin2, Survivin, Ku70, and XRCC6 levels suggests radiation inhibits the Wnt canonical pathway and failure in DNA repair mechanisms in a coupled manner. An increase in Bax, Bcl2, and caspase3 suggests apoptosis activation followed by the decreased expression of enzymatic antioxidants. Conclusion Controlled several interlinked such as the Wnt canonical pathway, NHEJ pathway, and intrinsic apoptotic pathway execute when the whole body is exposed to radiation. These pathways decide the cell fate whether it will survive or will go to apoptosis which may further be used in a study to counterpart and better comprehend medication focus on radiation treatment

Glyphosate decreases bovine oocyte quality by inducing oxidative stress and apoptosis.

Glyphosate is a universal herbicide with genital toxicity, but the effect of glyphosate on oocytes has not been reported. This study aimed to evaluate the effect of glyphosate (0, 10, 20, 50 and 100 mM) on bovine oocyte in vitro maturation. We showed that 50 mM glyphosate adversely affects the development of bovine oocytes. Exposure of oocytes to 50 mM glyphosate caused an abnormal reduction in oxidative (redox) levels compared with that in the control group, with a significantly higher reactive oxide species level (P < 0.05) and significantly lower glutathione (GSH) expression (P < 0.05). Additionally, the mRNA levels of antioxidant genes (SOD1, SOD2, SIRT2, SIRT3) and the mitochondrial membrane potential (MMP) were significantly reduced (P < 0.05). Furthermore, treatment with 50 mM glyphosate-induced apoptosis, and the mRNA levels of the apoptotic genes Caspase-3 and Caspase-4 were significantly higher than those in the control group (P < 0.05); however, the mRNA level of BAX was significantly higher than that in the control group (P < 0.01). Additionally, the mRNA levels of the anti-apoptotic genes Survivin and BCL-XL were significantly lower than those in the control group (P < 0.05), and oocyte quality was adversely affected. Together, our results confirmed that glyphosate impairs the quality of oocytes by promoting abnormal oocyte redox levels and apoptosis.

Matrine induces cell cycle arrest and apoptosis in hepatocellular carcinoma cells via miR-122 mediated CG1/livin/survivin signal axis

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To study the impact of matrine on cell cycle and apoptotic changes in hepatoma cells, and the mechanism involved.&#x0D; Methods: Human hepatoma cell line HepG2 was treated with different concentrations of matrine. The blank control cells were maintained in 1640 medium only. The influence of matrine on proliferative ability was determined with 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) method. Flow cytometry was used to determine its effect on cell cycle and apoptosis; RT-PCR (reverse transcription-polymerase chain reaction) was applied to assay the mRNA expressions of miR-122, cyclin G1 (CG1), livin and survivin mRNA, while the protein expressions of CG1, livin and surviving were assayed by Western blotting.&#x0D; Results: Matrine time- and dose-dependently suppressed the proliferative capacity of the cells. At a concentration of 0.5 mg/mL, matrine had no significant effect on the cell cycle. However, 1.0 mg/mL matrine blocked the cell cycle in G1 phase, while 1.5 mg/mL matrine blocked HepG2 cells in G2/M phase (p &lt; 0.05). Moreover, matrine induced apoptosis in HepG2 cells, and markedly downregulated the expressions of miR-122 concentration- time-reliantly (p &lt; 0.05). In addition, matrine markedly and concentration-dependently reduced mRNA and protein expression levels of CG1, livin and survivin, with the strongest inhibitory effect at a level of 1.5 mg/mL.&#x0D; Conclusion: Matrine induces cell cycle block and apoptotic changes in hepatoma cells through a mechanism related to regulation of the CG1/livin/survivin signal axis mediated by miR-122. Matrine may be a potential treatment for liver cancer. However, clinical trials are needed to confirm this potential.&#x0D; &#x0D; &#x0D; &#x0D;

Sanguinarine Reverses Pulmonary Vascular Remolding of Hypoxia-Induced PH via Survivin/HIF1α-Attenuating Kv Channels.

Background: Similarities in the biology of pulmonary hypertension and cancer suggest that anticancer therapies, such as sanguinarine, may also be effective in treating pulmonary hypertension. This, along with underlying biochemical pathways, is investigated in this study. Methods: Rats were subjected to 4-week hypoxia (or control) with or without sanguinarine treatment. In addition, pulmonary artery smooth muscle cells (PASMCs) were examined after 24–48 h hypoxia (with normoxic controls) and with or without sanguinirine. Pulmonary artery pressures and plasma survivin levels were measured in vivo. Ex vivo tissues were examined histologically with appropriate staining. mRNA and protein levels of survivin, HIF-1α, TGFb1, BMPR2, Smad3, P53, and Kv 1.2, 1.5, 2.1 were determined by real-time PCR and Western blot in PASMCs and distal PAs tissue. PASMC proliferation and changes of TGFb1 and pSmad3 induced by sanguinarine were studied using MTT and Western blot. Electrophysiology for Kv functions was measured by patch-clamp experiments. Results: Four-week hypoxia resulted in an increase in serum survivin and HIF-1α, pulmonary artery pressures, and pulmonary vascular remodeling with hypertrophy. These changes were all decreased by treatment with sanguinarine. Hypoxia induced a rise of proliferation in PASMCs which was prevented by sanguinarine treatment. Hypoxic PASMCs had elevated TGFb1, pSmad3, BMPR2, and HIF1α. These increases were all ameliorated by sanguinarine treatment. Hypoxia treatment resulted in reduced expression and function of Kv 1.2, 1.5, 2.1 channels, and these changes were also modulated by sanguinarine. Conclusion: Sanguinarine is effective in modulating hypoxic pulmonary vascular hypertrophy via the survivin pathway and Kv channels.

Cytotoxic activity related to survivin mRNA levels by Combretum quadrangulare Kurz extract against liver and breast cancer cells

Liver cancer and breast cancer have high death rate and increased rapidly in recent years in Vietnam and over the world. Currently, the study of bioactive compounds isolated from plants for killing cancer cells is of interest. Herein, the study aims to identify the cytotoxicity using MTT assay of Combretum quadrangulare Kurz fractions against MCF-7 cells. RT-qPCR method was used to determine the survivin mRNA levels in P12 fraction-treated HepG2 and MCF-7 in comparison to DMSO-treated cells. The results showed that P12 fraction with IC50 of 20.9 ± 0.8 µg/mL was the best cytotoxicity on MCF-7 cells amongst P fractions. P12 fraction exhibited the apoptotic effect on HepG2 and MCF-7 cells by down-regulating survivin expression levels. Thus, P12 fraction was promising for the next research for the impact of fractions assessment in vivo or bioactive compounds isolation.

PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing.

PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs.

Broad-Spectrum Polymeric Nanoquencher as an Efficient Fluorescence Sensing Platform for Biomolecular Detection.

Colloidal inorganic nanostructures (metal, carbon, and silica) have been widely used as "nanoquenchers" for construction of nanosensors; however, inherent drawbacks such as insufficient fluorescence quenching efficiency, false positive signals, and uncertain long-term cytotoxicity have limited their further utility. Herein, by taking advantages of polymeric nanoparticles (PNPs) in terms of high loading capacity, facile surface modification chemistry, and good biocompatibility, we report a broad-spectrum (400-750 nm) polymeric fluorescence-quenching platform for sensor fabrication. Our newly developed polymeric nanoquenchers (qPNPs) were constructed by concurrently encapsulating various alkylated black-hole quenchers into nanoparticles made of poly(methyl methacrylate-co-methacrylic acid) and were found to have an excellent fluorescence quenching effect (>400-fold) on common fluorophores (FAM, TMR, and Cy5) together with high stability under physiological conditions. As a proof of concept, the feasibility of these qPNPs for fluorescence sensing was validated by successful construction of two nanosensors (FAMDEVD@qPNP and Cy5SurC@qPNP), which could be used as promising nanosensors for live-cell imaging of the apoptosis-related protease caspase-3 and cancer-related survivin mRNA, respectively. As expected, in the FAM channel, the FAMDEVD@qPNP showed fast and selective fluorescence responses toward caspase-3 in buffers and could be used to image the activation of drug-induced endogenous caspase-3. In the Cy5 channel, the Cy5SurC@qPNP could be used to distinguish normal cells (MCF10A) from cancer cells (HeLa) by quantitatively detecting the endogenous survivin mRNA level. It could be further used to monitor changes in the endogenous survivin mRNA expression levels in drug-treated HeLa cells. Altogether, by virtue of their high quencher loading and broad-spectrum quenching efficiency and good signal-to-background ratio, these qPNPs might be particularly attractive alternatives to other conventional nanoquenchers for the construction of more complex biosensors in the future.

Wogonin Stimulation of Cell Death and Reducing Survivin in MDM-MB231 Breast Tumors

Background: Wogonin as a flavonoid compound is known for its anticancer effect through growth control, differentiation, and apoptosis. This study investigates the possible activity of wogonin in inducing cell death of breast tumors MDA-MB231. Materials and Methods: The level of cell proliferation was evaluated by MTT assay. Cycle analysis and the apoptotic rate were assessed by flow cytometry. mRNA level of Bax, Bcl-2, p53, and survivin was evaluated by real-time PCR. Western blot was utilized to assert the relative protein expression. Results: Wogonin inhibits the proliferation of MDA-MB231 over time, and in particular, wogonin can induce the arrest of phase G1 of MDA-MB231 cells. The apoptosis of wogonin was related to a remarkable decrease in Bcl-2 and survivin and an increment in p53 and Bax. Wogonin also significantly elevated the active apoptotic forms of caspase-3, -8, -9. Z-DEVD-FMK, a specific inhibitor of caspase-3, remarkably inhibits cellular apoptosis caused by wogonin. Wogonin suppresses PI3K/Akt phosphorylation and ERK-derived phosphorylation. PD98059, a specific ERK inhibitor, significantly blocked the apoptosis caused by wogonin. Also, LY294002, a specific suppressor of PI3K, remarkably elevates the cellular apoptosis caused by wogonin. Various studies demonstrated that LY294002 not only modulates the expression of the survivin gene alone but also enhances the suppression of the expression of survivin with wogonin. Conclusion: The apoptotic role of wogonin is observed by the triggering caspases and ERK and is associated with the suppressive pathways of the PI3K/Akt/survivin signal in the MDA-MB231 cells.

Coexpression of Smac/DIABLO and Estrogen Receptor in breast cancer.

Smac/DIABLO is a proapoptotic protein deregulated in breast cancer, with a controversial role as a tumor marker, possibly due to a lack of correlative mRNA and protein analyses. To investigate the association of Smac/DIABLO gene and protein levels with clinical variables in breast cancer patients. Smac/DIABLO mRNA expression was analyzed by qPCR in 57 frozen tissues, whereas protein levels were assessed by immunohistochemistry in 82 paraffin-embedded tissues. Survivin mRNA levels were also measured. In vitro assays were performed to investigate possible regulators of Smac/DIABLO. Higher levels of Smac/DIABLO mRNA and protein were found in estrogen receptor (ER)-positive samples (p= 0.0054 and p= 0.0043, respectively) in comparison to ER-negative tumors. A negligible positive association was found between Smac/DIABLO and survivin expression. In vitro assays showed that Smac/DIABLO is not regulated by ER and, conversely, it does not participate in ER expression modulation. mRNA and protein levels of Smac/DIABLO were increased in ER-positive breast tumors in comparison with ER-negative samples, although the mechanism of this regulation is still unknown. Public databases showed a possible clinical relevance for this association.

The Combination of Zerumbone and 5-FU: A Significant Therapeutic Strategy in Sensitizing Colorectal Cancer Cells to Treatment

Objectives. Chemotherapy is considered to be essential in the treatment of patients with colorectal cancer (CRC), but drug resistance reduces its efficacy. Many patients with advanced CRC eventually show resistance to 5-fluorouracil (5-FU) therapy. Synergistic and potentiating effects of combination therapy, using herbal and chemical drugs, can improve patients’ response. Zerumbone (ZER), which is derived from ginger, has been studied for its growth inhibitory function in various types of cancer. Methods. The cytotoxic effects of ZER and 5-FU alone and their combination, on the SW48 and HCT-116 cells, were examined, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The mRNA and protein levels of β-catenin, survivin, and vimentin were measured in treated CRC cells, using qRT-PCR and western blot. Colony formation assay, scratch test, and flow cytometry were performed to detect the changes of proliferation, migration, and apoptosis. Key Findings. In HCT-116- and SW48-treated cells, the proliferation, the gene and protein expression levels of the markers, the migration, the colony formation, and the survival rates were all significantly reduced compared to the control groups, and the sharpest decline was observed in the 5-FU+ZER treatment groups. Conclusions. Combination therapy has shown promising results in CRC cells, especially in drug-resistant cells.

Preparation Optimization of Bovine Serum Albumin Nanoparticles and Its Application for siRNA Delivery.

BackgroundsiRNA brings hope for cancer therapy. However, there are many obstacles for application of siRNA in clinical. Because of the excellent biocompatibility, non-toxicity and non-immunogenicity of bovine serum albumin (BSA), BSA-based nanoparticles have been widely designed as a drug carrier system.MethodsThe optimal formula for BSA NPs preparation was investigated by central composite design response surface methodology (CCD-RSM), BSA-based survivin-siRNA delivery system (BSA NPs/siRNA) was characterized by dynamic light scattering, atomic force microscope, transmission electron microscope and Bradford method. The in vitro anti-tumor effect and mechanism of BSA NPs were investigated by confocal microscopic imaging, MTT assay, RT-qPCR and ELISA analysis. Moreover, the anti-tumor effect, distribution and biosafety of BSA NPs were studied in vivo.ResultsThe optimal formula for BSA NPs was settled to be 20 mg/mL for BSA concentration, 9 for pH value, 136% for crosslinking degree and 1.6 mL/min for speed of ethanol addition. BSA NPs/siRNA could remain stable at 4°C for 4 weeks and protect siRNA from degradation by RNase A. Besides, BSA NPs/siRNA could maintain a sustained release of siRNA and promote the uptake of siRNA significantly. The survivin-mRNA level and the survivin-protein level were decreased by 55% ± 1.6% and 54% ± 1.6% separately. The in vivo tumor inhibition results suggested that the tumor inhibition rate of BSA NPs/siRNA-treated group was 54% ± 12% and was similar with that of DOX-treated group (57% ± 9.2%, P > 0.05). The biosafety results confirmed that BSA NPs/siRNA could not induce significant damages to the main organs and blood in vivo.ConclusionThese results demonstrated that CCD-RSM was an effective tool for preparation analysis, and the BSA NPs/siRNA was a promising system for siRNA-based gene therapy.

Inhibitor of Multi-cyclin-dependent Kinases (AT7519) Reduced Survival of U937 Leukemic Cells and Enhanced Anti-leukemic Effect of Vincristine: A Highlight to CDK Inhibition Efficacy in Acute Leukemia

Background: The conservative character of the cell cycle outlined that any dysregulation in the regulatory components of this process in normal cells opens a gate toward neoplastic transformation. Objectives: Given the critical role of cyclin-dependent kinases (CDKs) in cancer pathogenesis and based on their frequent aberrancy in human leukemia, the present study aimed at evaluating the suppressive effect of a multi-CDK inhibitor AT7519 on acute myeloid leukemia-derived U937 cells. Methods: To assess the anti-leukemic effects of the inhibitor on acute myeloid leukemia (AML) cells, we used MTT and trypan blue assays. Flow cytometric analysis and q-RT-PCR were also applied to evaluate the impact of AT7519 on cell cycle and apoptosis. Results: The results suggested that suppression of CDK in U937 cells hampered the proliferation of leukemic cells through a G2/M arrest mediated by p21 gene. Additionally, the anti-survival impact of AT7519 on these cells was shown to be along with the apoptosis initiation not only through the increment of pro-apoptotic gene expression but also through diminishing the mRNA levels of both Pin1 and Survivin. Notably, the potent anti-leukemic property of this agent has become more prominent when we found that the blockage of CDKs in AML cells could synergize with the cytotoxic effect of vincristine (VCR). To the best of our knowledge, little is known about the molecular mechanisms of resistance to AT7519 and we proposed that the effectiveness of this agent was partially attenuated through either c-Myc or autophagy activation in U937 cells. Conclusions: This study suggests that the pharmacological targeting of CDKs could probably unwind the complexity of therapeutic obstacles on the way of acute leukemia, either in the context of mono- or combined-modal strategy.