Transfection of Unmodified MicroRNA Using Monolayer-Coated Au Nanoparticles as Gene-Delivery Vehicles.

This article describes a monolayer-coated gold nanoparticle-based transfection system for the delivery of microRNA (miRNA) into human osteosarcoma (HOS) cells. Two distinct ammonium-terminated adsorbates were used in this study, which provided a platform for ionic bonding of the miRNA onto gold nanoparticles (AuNPs). The custom-designed monolayer-coated gold nanoparticles were characterized by dynamic light scattering, gel mobility shift assay, transmission electron microscopy, ultraviolet-visible spectrometry, zeta potential, and X-ray photoelectron spectroscopy. The miRNA-loaded gold nanoparticles were transfected, and the level of intracellular miRNA delivered and taken up by cells was measured by Taqman qPCR. The overall analysis indicated a successful delivery of miRNA into the HOS cells at an ∼11,000-fold increase compared to nontreated cells.

2,4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone from Cleistocalyx nervosum var. paniala seeds attenuated the early stage of diethylnitrosamine and 1,2-dimethylhydrazine-induced colorectal carcinogenesis.

BACKGROUND: Dichloromethane extract of Cleistocalyx nervosum var. paniala seeds exhibited an anticarcinogenicity against chemically-induced the early stages of carcinogenesis in rats. This study aimed to identify anticarcinogenic compounds from C. nervosum seed extract (CSE). METHODS: Salmonella mutation assay was performed to determine mutagenicity and antimutagenicity of partially purified and purified compounds of CSE. The anticarcinogenic enzyme-inducing activity was measured in Hepa1c1c7. Moreover, the anticancer potency was examined on various human cancer cell lines. The anticarcinogenicity of DMC was investigated using dual-organ carcinogenicity model. The number of preneoplastic lesions was evaluated in the liver and colon. The inhibitory mechanisms of DMC on liver- and colorectal carcinogenesis were investigated. RESULTS: Six partially purified fractions (MK1 - MK6) and purified compounds, including 2,4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) and hariganetin, were obtained from CSE. Among these fractions, MK4 and DMC presented the greatest antimutagenicity against indirect mutagens in bacterial model. Moreover, MK5 possessed an effective anticarcinogenic enzyme inducer in Hepa1c1c7. The MK4, DMC and CSE showed greater anticancer activity on all cell lines and exhibited the most effective toxicity on colon cancer cells. Furthermore, DMC inhibited the formation of colonic preneoplastic lesions in carcinogens-treated rats. It reduced PCNA-positive cells and frequency of BCAC in rat colon. DMC also enhanced the detoxifying enzyme, GST, in rat livers. CONCLUSIONS: DMC obtained from CSE may be a promising cancer chemopreventive compound of colorectal cancer process in rats. It could increase detoxifying enzymes and suppress the cell proliferation process resulting in prevention of post-initiation stage of colorectal carcinogenesis.

Pyrogallol from Spirogyra neglecta Inhibits Proliferation and Promotes Apoptosis in Castration-Resistant Prostate Cancer Cells via Modulating Akt/GSK-3ß/ß-catenin Signaling Pathway.

Castration-resistant prostate cancer (CRPC) is an advanced form of prostate cancer associated with poor survival rates. The high proliferation and metastasis rates have made CRPC one of the most challenging types of cancer for medical practitioners and researchers. In this study, the anti-cancer properties and inhibition of CRPC progression by S. neglecta extract and its active constituents were determined using two CRPC cell lines, DU145 and PC3. The ethyl acetate fraction of S. neglecta (SnEA) was obtained using a solvent-partitioned extraction technique. The active constituents of SnEA were then determined using the HPLC technique, which showed that SnEA mainly contained syringic acid, pyrogallol, and p-coumaric acid phenolic compounds. After the determination of cytotoxic properties using the SRB assay, it was found that pyrogallol, but not the other two major compounds of SnEA, displayed promising anti-cancer properties in both CRPC cell lines. SnEA and pyrogallol were then further investigated for their anti-proliferation and apoptotic induction properties using propidium iodide and Annexin V staining. The results showed that SnEA and pyrogallol inhibited both DU145 and PC3 cell proliferation by inducing cell cycle arrest in the G0/G1 phase and significantly decreased the expression of cell cycle regulator proteins (cyclin D1, cyclin E1, CDK-2, and CDK-4, p < 0.001). SnEA and pyrogallol treatments also promoted apoptosis in both types of CRPC cells through significantly downregulating anti-apoptotic proteins (survivin, Bcl-2, and Bcl-xl, p < 0.001) and upregulating apoptotic proteins (cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP-1, p < 0.001). Mechanistic study demonstrated that SnEA and pyrogallol inactivated the Akt signaling pathway leading to enhancement of the active form of GSK-3ß in CRPC cell lines. Therefore, the phosphorylation of ß-catenin was increased, which caused degradation of the protein, resulting in a downregulation of ß-catenin (unphosphorylated form) transcriptional factor activity. The current results reflect the potential impact of S. neglecta extract and pyrogallol on the management of castration-resistant prostate cancer.

Hydrosoluble Perylene Monoimide-Based Telomerase Inhibitors with Diminished Cytotoxicity.

Telomerase is essential for the immortality characteristics of most cancers. Telomerase-specific inhibitors should render cancer cells to replicative senescence without acute cytotoxicity. Perylene-based G-quadruplex (G4) ligands are widely studied as telomerase inhibitors. Most reported perylene-based G4 ligands are perylene diimides (PDIs), which often suffer from self-aggregation in aqueous solutions. Previously, we found that PM2, a perylene monoimide (PMI), exhibited better solubility, G4 binding affinity, and telomerase inhibition than PIPER, the prototypic PDI. However, the acute cytotoxicity of PM2 was about 20-30 times more than PIPER in cancer cells. In this report, we replaced the piperazine side chain of PM2 with ethylenediamine to yield PM3 and replaced the N,N-diethylethylenediamine side chain of PM2 with the 1-(2-aminoethyl) piperidine to yield PM5. We found that asymmetric PMIs with two basic side chains (PM2, PM3, and PM5) performed better than PIPER (the prototypic PDI), in terms of hydrosolubility, G4 binding, in vitro telomerase inhibition, and suppression of human telomerase reverse transcriptase (hTERT) expression and telomerase activity in A549 cells. However, PM5 was 7-10 times less toxic than PM2 and PM3 in three cancer cell lines. We conclude that replacing the N,N-diethylethylenediamine side chain with the 2-aminoethylpiperidine on PMIs reduces the cytotoxicity in cancer cells without impacting G4 binding and telomerase inhibition. This study paves the way for synthesizing new PMIs with drug-like properties for selective telomerase inhibition.

Telomere shortening correlates with disease severity in hemoglobin H disease patients.

Hemoglobin H (Hb H) disease is the most significant health problem of the α-thalassemia syndromes. The Hb disease patients are categorized based on their genotype to deletional and nondeletional, with the latter genotype presents the more severe clinical symptoms. Since telomere length is an indicator of biological aging and health, we hypothesized that telomere length could reflect Hb H disease's severity. In this study, we recruited 48 deletional and 47 nondeletional Hb H disease patients, along with 109 normal controls, for telomere length assessment. The leukocyte telomere length was assessed by monochromatic multiplex real-time PCR and reported as the telomere to single-copy gene (T/S) ratio. When telomere length was adjusted for age, the analysis of covariance between the control and the two Hb H disease groups revealed no significant difference. However, the telomere shortening rate was more rapid in the nondeletional Hb H disease group than those of the control and deletional Hb H disease groups. Gender analysis found that male patients have a significantly lower T/S ratio than females in the nondeletional group but not in the control and deletional groups. In the two disease groups, the T/S ratio was not influenced by ferritin level or transfusion burden but was positively correlated with the absolute reticulocyte count.

Superiority of an Asymmetric Perylene Diimide in Terms of Hydrosolubility, G-Quadruplex Binding, Cellular Uptake, and Telomerase Inhibition in Prostate Cancer Cells.

Perylene diimide (PDI) derivatives have been studied as G-quadruplex ligands that suppress telomerase activity by facilitating G-quadruplex formation of telomeric DNA and the hTERT promoter. PIPER, the prototypical PDI, reduces telomerase activity in lung and prostate cancer cells, leading to telomere shortening and cellular senescence of these cells. However, PIPER suffers from poor hydrosolubility and the propensity to aggregate at neutral pH. In this report, we synthesized a new asymmetric PDI, aPDI-PHis, which maintains one N-ethyl piperidine side chain of PIPER and has histidine as another side chain. The results show that aPDI-PHis is superior to its symmetric counterparts, PIPER and PDI-His, in terms of hydrosolubility, G-quadruplex binding, cellular uptake, and telomerase inhibition in prostate cancer cells. These results suggest that one N-ethyl piperidine side chain of PDI is sufficient for G-quadruplex binding, while another side chain can be tuned to elicit desirable properties. These findings might lead to better PDIs for use as anticancer drugs.

Inactivation of AKT/NF­κB signaling by eurycomalactone decreases human NSCLC cell viability and improves the chemosensitivity to cisplatin.

The high activation of protein kinase B (AKT)/nuclear factor­κB (NF­κB) signaling has often been associated with the induction of non­small cell lung cancer (NSCLC) cell survival and resistance to cisplatin, which is one of the most widely used chemotherapeutic drugs in the treatment of NSCLC. The inhibition of AKT/NF­κB can potentially be used as a molecular target for cancer therapy. Eurycomalactone (ECL), a quassinoid from Eurycoma longifolia Jack, has previously been revealed to exhibit strong cytotoxic activity against the human NSCLC A549 cell line, and can inhibit NF­κB activity in TNF­α­activated 293 cells stably transfected with an NF­κB luciferase reporter. The present study was the first to investigate whether ECL inhibits the activation of AKT/NF­κB signaling, induces apoptosis and enhances chemosensitivity to cisplatin in human NSCLC cells. The anticancer activity of ECL was evaluated in two NSCLC cell lines, A549 and Calu­1. ECL decreased the viability and colony formation ability of both cell lines by inducing cell cycle arrest and apoptosis through the activation of pro­apoptotic caspase­3 and poly (ADP­ribose) polymerase, as well as the reduction of anti­apoptotic proteins Bcl­xL and survivin. In addition, ECL treatment suppressed the levels of AKT (phospho Ser473) and NF­κB (phospho Ser536). Notably, ECL significantly enhanced cisplatin sensitivity in both assessed NSCLC cell lines. The combination treatment of cisplatin and ECL promoted cell apoptosis more effectively than cisplatin alone, as revealed by the increased cleaved caspase­3, but decreased Bcl­xL and survivin levels. Exposure to cisplatin alone induced the levels of phosphorylated­AKT and phosphorylated­NF­κB, whereas co­treatment with ECL inhibited the cisplatin­induced phosphorylation of AKT and NF­κB, leading to an increased sensitization effect on cisplatin­induced apoptosis. In conclusion, ECL exhibited an anticancer effect and sensitized NSCLC cells to cisplatin through the inactivation of AKT/NF­κB signaling. This finding provides a rationale for the combined use of chemotherapy drugs with ECL to improve their efficacy in NSCLC treatment.

Leukocyte telomere length in patients with transfusion-dependent thalassemia.

BACKGROUND: Thalassemia is a hereditary hemolytic anemia with a severity ranging from mild, non-transfusion dependent to severe chronic anemia requiring lifelong transfusion. Transfusional iron overload is a major complication in patients with transfusion-dependent thalassemia (TDT). Telomeres are sequences of nucleotides forming the end caps of chromosomes that act as a DNA repair system. Iron overload in thalassemia can cause increased oxidative stress which leads to cellular damage and senescence. This may result in telomere length shortening. The degree of telomere length shortening may reflect the severity of thalassemia. METHODS: This research aimed to study the leukocyte telomere length in patients with TDT in comparison to non-thalassemic individuals and to identify the clinical and laboratory parameters that are associated with telomere length. We conducted a cross-sectional study in patients with TDT aged ≥18 years. Leukocyte telomere length was measured by real-time quantitative PCR. RESULTS: Sixty-five patients with TDT were enrolled onto the study. There were 37 female patients (54.4%). The median age was 27 (18-57) years, and mean pre-transfusion hemoglobin level was 7.1 (± 1.07) g/dL. The mean telomere to single copy gene (T/S) ratios of patients with TDT and the controls were 0.72 ± 0.18 and 0.99 ± 0.25, respectively (p < 0.0001). There was a significant correlation between the T/S ratio and age (p = 0.0002), and hemoglobin level (p = 0.044). There was no correlation between telomere length and other factors. CONCLUSIONS: Our study showed that TDT patients had shorter leukocyte telomere length compared with controls. Leukocyte telomere shortening in TDT was an aging-dependent process and associated with lower hemoglobin level.

Telomerase Inhibition, Telomere Shortening, and Cellular Uptake of the Perylene Derivatives PM2 and PIPER in Prostate Cancer Cells.

Prostate cancer is the second most common cancer among men worldwide, and it is ranked first in the United States and Europe. Since prostate cancer is slow-growing, active surveillance for low-risk cancer has been increasingly supported by various guidelines. Most prostate cancers reactivate telomerase to circumvent the replicative senescence caused by the end replication problem; therefore, telomerase inhibition is potentially useful for the suppression of prostate cancer progression during this active surveillance or for the prevention of cancer recurrence after conventional therapies. In this study, we demonstrated that the perylene derivatives, PM2 and PIPER, could suppress human telomerase reverse transcriptase (hTERT) expression and telomerase activity in the short-term treatment of androgen-dependent prostate cancer cell line LNCaP and the androgen-independent prostate cancer cell line PC3 prostate cancer cells. Long-term treatment with subcytotoxic doses of these compounds in both prostate cancer cells showed telomere shortening and a significant increase in senescent cells. Although the acute cytotoxicity of PM2 was about 30 times higher than that of PIPER in both prostate cancer cells, the cellular uptake of both compounds was comparable as determined by flow cytometry and fluorescent microscopy.

Short Telomere Length as a Biomarker Risk of Lung Cancer Development Induced by High Radon Levels: A Pilot Study.

Long-term exposure to radon has been determined to be the second leading cause of lung cancer after tobacco smoking. However, an in-depth study of this topic has not been explicitly carried out in Chiang Mai (Thailand). This paper presents the results of an indoor radon level measurement campaign in dwellings of Chiang Mai using total of 110 detectors (CR-39) during one year. The results show that the average radon levels varied from 35 to 219 Bq/m³, with an overall average of 57 Bq/m³. The finding also shows that the average value is higher than the global average value of 39 Bq/m³. In addition, to examine the cause of lung cancer development among people with risk of chronic exposure to radon during their lifetime, 35 non-smoker lung cancer patients and 33 healthy nonsmokers were analyzed for telomere length. As expected, telomere length was significantly shorter in lung cancer patients than in healthy nonsmokers. Among healthy nonsmokers, the telomere length was significantly shorter in a high radon group than in an unaffected low radon group. To the best of our knowledge, our research provides the first attempt in describing the shortened telomeres in areas with high levels of environmental radon that might be related to lung cancer development.

Ginger Extract Promotes Telomere Shortening and Cellular Senescence in A549 Lung Cancer Cells.

Replicative senescence, which is caused by telomere shortening from the end replication problem, is considered one of the tumor-suppressor mechanisms in eukaryotes. However, most cancers escape this replicative senescence by reactivating telomerase, an enzyme that extends the 3'-ends of the telomeres. Previously, we reported the telomerase inhibitory effect of a crude Zingiber officinale extract (ZOE), which suppressed hTERT expression, leading to a reduction in hTERT protein and telomerase activity in A549 lung cancer cells. In the present study, we found that ZOE-induced telomere shortening and cellular senescence during the period of 60 days when these A549 cells were treated with subcytotoxic doses of ZOE. Using assay-guided fractionation and gas chromatography/mass spectrometry analysis, we found that the major compounds in the active subfractions were paradols and shogaols of various chain lengths. The results from studies of pure 6-paradol and 6-shogaol confirmed that these two compounds could suppress hTERT expression as well as telomerase activity in A549 cells. These results suggest that these paradols and shogaols are likely the active compounds in ZOE that suppress hTERT expression and telomerase activity in these cells. Furthermore, ZOE was found to be nontoxic and had an anticlastogenic effect against diethylnitrosamine-induced liver micronucleus formation in rats. These findings suggest that ginger extract can potentially be useful in dietary cancer prevention.

Prevalence of α-thalassaemia genotypes in pregnant women in northern Thailand.

BACKGROUND & OBJECTIVES: Alpha-thalassaemias are genetic disorders with high prevalence in northern Thailand. However, common genotypes and current data on the prevalence of α-thalassaemias have not been reported in this region. Therefore, the objective of the present study was to determine the prevalence of α-thalassaemia genotypes in pregnant women in northern Thailand. METHODS: Genomic DNA was extracted from blood samples of pregnant women who came to Maharaj Nakorn Chiang Mai University Hospital during July 2009 to 2010. The common deletion and point mutation genotypes of α-thalassaemia were evaluated by gap- polymerase chain reaction (PCR) and PCR with restriction fragment length polymorphism (RFLP). RESULTS: Genotypes of 638 pregnant women were: 409 samples (64.11%) being normal subjects (αα/αα) and 229 samples (35.89%) with α-thalassaemias. these 229 samples could be classified into deletional HbH disease (--SEA/-α3.7) for 18 samples (2.82%); heterozygous α0-thalassaemia --SEA type (--SEA/αα)) for 78 (12.23%); heterozygous α+-thalassaemia - α3.7 type (-α3.7/αα) for 99 (15.52%); homozygous α+-thalassaemia - α3.7 type (-α3.7/- α3.7) for five (0.78%); heterozygous α+-thalassaemia - α4.2 type (-α4.2/αα) for two (0.31%); and heterozygous HbCS (αCSα/αα) for 27 (4.23%) cases. INTERPRETATION & CONCLUSIONS: The prevalence of α-thalassaemias in pregnant women in northern Thailand was high. This finding supports the implementation of the prevention and control of this common genetic disorder by screening for α-thalassaemia genotypes.

Curcuminoid derivatives enhance telomerase activity in an in vitro TRAP assay.

The length of telomeres controls the life span of eukaryotic cells. Telomerase maintains the length of telomeres in certain eukaryotic cells, such as germline cells and stem cells, and allows these cells to evade replicative senescence. Here, we report for the first time a number of curcuminoid derivatives that enhance telomerase activity in an in vitro TRAP assay. A preliminary analysis of structure-activity relationships found that the minimal requirement for this enhanced telomerase activity is a curcuminoid core with at least one n-pentylpyridine side chain, while curcuminoids with two such side chains exhibit even greater activity. The finding here might lead to a new class of telomerase activators that act directly or indirectly on telomerase, rather than through the reactivation of the telomerase reverse transcriptase (TERT) gene associated with other telomerase activators found in the literature.

Telomere shortening and cell senescence induced by perylene derivatives in A549 human lung cancer cells.

Cancer cells evade replicative senescence by re-expressing telomerase, which maintains telomere length and hence chromosomal integrity. Telomerase inhibition would lead cancer cells to senesce and therefore prevent cancer cells from growing indefinitely. G-quadruplex ligands can attenuate telomerase activity by inducing G-quadruplex formation at the 3'-overhang of telomere and at the human telomerase reverse transcriptase (hTERT) promoter; the former prevents telomerase from accessing the telomere, and the latter acts as a transcriptional silencer. The present investigation found that perylene derivatives PM2 and PIPER induced G-quadruplex formation from both telomeric DNA and the hTERT promoter region in vitro. Further, TRAP assay showed that these compounds inhibited telomerase in a dose-dependent manner. When A549 human lung cancer cells were treated with these compounds, hTERT expression was down-regulated. Moreover, the crude protein extract from these treated cells exhibited less telomerase activity. In the long-term treatment of A549 lung cancer cells with sub-cytotoxic dose of these perylenes, telomere shortening, reduction of cell proliferation and tumorigenicity, and cell senescence were observed. The results of this study indicate that perylene derivatives warrant further consideration as effective agents for cancer therapy.

Aristolactam-type alkaloids from Orophea enterocarpa and their cytotoxicities.

A new aristolactam, named enterocarpam-III (10-amino-2,3,4,6-tetramethoxy phenanthrene-1-carboxylic acid lactam, 1) together with the known alkaloid stigmalactam (2), were isolated from Orophea enterocarpa. Their structures were elucidated on the basis of interpretation of their spectroscopic data. Compounds 1 and 2 exhibited significant cytotoxicities against human colon adenocarcinoma (HCT15) cell line with IC(50) values of 1.68 and 1.32 µM, respectively.

Down-regulation of the human VEGF gene expression by perylene monoimide derivatives.

The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a guanine-rich strand that can act as a transcriptional silencer by forming an intramolecular G-quadruplex. In this study, we compared two perylene monoimide derivatives, PM1 and PM2, with the well-studied perylene diimide derivative, PIPER, and the well-studied porphyrin derivative, TmPyP(4), with regard to G-quadruplex formation, G-quadruplex binding selectivity, and human VEGF gene silencing in A549 lung cancer cells. The results show that these perylene derivatives can preferentially induce intramolecular G-quadruplex formation from a duplex containing the VEGF G-quadruplex motif in vitro. Incubating A549 lung cancer cells with these perylene derivatives, especially PM2, led to the reduction of both VEGF mRNA and VEGF protein. This study might provide the foundation for the rational design and development of new perylene derivatives as effective anti-angiogenesis agents for cancer therapy.

Ginger extract inhibits human telomerase reverse transcriptase and c-Myc expression in A549 lung cancer cells.

The rhizome of ginger (Zingiber officinale Roscoe) has been reputed to have many curative properties in traditional medicine, and recent publications have also shown that many agents in ginger possess anticancer properties. Here we show that the ethyl acetate fraction of ginger extract can inhibit the expression of the two prominent molecular targets of cancer, the human telomerase reverse transcriptase (hTERT) and c-Myc, in A549 lung cancer cells in a time- and concentration-dependent manner. The treated cells exhibited diminished telomerase activity because of reduced protein production rather than direct inhibition of telomerase. The reduction of hTERT expression coincided with the reduction of c-Myc expression, which is one of the hTERT transcription factors; thus, the reduction in hTERT expression might be due in part to the decrease of c-Myc. As both telomerase inhibition and Myc inhibition are cancer-specific targets for cancer therapy, ginger extract might prove to be beneficial as a complementary agent in cancer prevention and maintenance therapy.

The influence of pH on the G-quadruplex binding selectivity of perylene derivatives.

Three new perylene derivatives with branched ionizable side chains were synthesized, and their G-quadruplex binding specificities were compared by spectroscopic and electrophoretic analysis with two well-studied G-quadruplex ligands: PIPER and TmPyP4. The value of pH and consequent charge formation and self-aggregation of these perylene derivatives influences not only the type of G-quadruplex formation, but also the G-quadruplex binding selectivity.

Well-ordered self-assembled monolayer surfaces can be used to enhance the growth of protein crystals.

A series of hydrophobic self-assembled monolayers (SAMs) was generated by the adsorption of undecanethiol, dodecanethiol, and octadecanethiol onto transparent gold-coated glass microscope slides. Protein crystallization trials using droplets deposited on the surfaces of the optically transparent SAMs were compared to those for which the droplets were deposited on the surfaces of conventional silanized glass microscope slides. For the five distinct proteins examined in the crystallization trials (i.e., lysozyme, alpha-lactalbumin, hemoglobin, thaumatin, and catalase), the SAMs generally afforded, (1) a faster rate of crystallization, (2) a larger crystal size; and (3) a broader range of crystallization conditions than that afforded by silanized glass. The greatest enhancements were observed with the highly ordered SAMs derived from octadecanethiol, which are evaluated here for the first time.