Cytotoxicity of juglone and thymoquinone against pancreatic cancer cells.

Juglone and thymoquinone are cytotoxic to pancreatic cancer cells. The aim of this study was to investigate, using an analysis of isobolograms, the type and degree of interactions between juglone and thymoquinone on MIA PaCa-2 pancreatic cancer cells. Cell viability was evaluated using the MTT assay. Cell death was determined by flow cytometry. The IC50 value for juglone and TQ in combination was found to be 24.75 µM, which was higher than juglone or TQ alone. Juglone alone killed Mia Paca-2 cells by ferroptosis. At concentrations where 10, 20 or 50% of cells were affected, there existed a moderate antagonistic relationship between juglone and TQ as indicated by the combination index (CI) value determined by the Compusyn software. At concentrations that affected 75% and 90% of cells, there were nearly an additive effect with CI value of 1.09249 and 0.92391, respectively. Moderate synergism was only seen at concentration where 95% of cells were affected, and the corresponding concentration of juglone and TQ at that combination was 40.90 µM and 511.19 µM, respectively.

An extract of Artemisia dracunculus L. stimulates insulin secretion from ß cells, activates AMPK and suppresses inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia dracunculus L. (Russian tarragon) is a perennial herb belonging to the family Compositae and has a history of medicinal use in humans, particularly for treatment of diabetes. AIM OF THE STUDY: In this study a defined plant extract from A. dracunculus L. (termed PMI-5011) is used to improve beta(ß) cells function and maintain ß cell number in pancreatic islets as an alternative drug approach for successful treatment of diabetes. MATERIALS AND METHODS: Mouse and human pancreatic beta cells were treated with defined plant extract of A. dracunculus L. (PMI-5011) to understand the mechanism(s) that influence beta cell function and ß cell number. RESULTS: We found that the PMI-5011 enhances insulin release from primary ß cells, isolated mouse and human islets and it maintains ß cell number. Insulin released by PMI-5011 is associated with the activation of AMP-activated protein kinase (AMPK), and protein kinase B (PKB). Furthermore, PMI-5011 suppresses LPS/INFγ-induced inflammation and inflammatory mediator(s) in macrophages. PMI-5011 inhibited Nitric oxide (NO) production and expression of inducible nitric oxide synthase (iNOS) at the protein level and also attenuated pro-inflammatory cytokine (IL-6) production in macrophages. CONCLUSION: PMI-5011 has potential therapeutic value for diabetes treatment via increasing insulin release from ß cells and decreases capacity of macrophages to combat inflammation.

Tunable cytotoxicity of rhodamine 6G via anion variations.

Chemotherapeutic agents with low toxicity to normal tissues are a major goal in cancer research. In this regard, the therapeutic activities of cationic dyes, such as rhodamine 6G, toward cancer cells have been studied for decades with observed toxicities toward normal and cancer cells. Herein, we report rhodamine 6G-based organic salts with varying counteranions that are stable under physiological conditions, display excellent fluorescence photostability, and more importantly have tunable chemotherapeutic properties. Our in vitro studies indicate that the hydrophobic compounds of this series allow production of nanoparticles which are nontoxic to normal cells and toxic to cancer cells. Furthermore, the anions, in combination with cations such as sodium, were observed to be nontoxic to both normal and cancer cells. To the best of our knowledge, this is the first demonstration that both the cation and anion play an extremely important and cooperative role in the antitumor properties of these compounds.

APOBEC3 inhibits DEAD-END function to regulate microRNA activity.

The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3'-untranslated region (3'-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3'-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3'-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3-/- mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function.

Lipophilic phosphonium-lanthanide compounds with magnetic, luminescent, and tumor targeting properties.

Multifunctional phosphonium-lanthanide compounds that simultaneously possess paramagnetism, luminescence, and tumor mitochondrial targeting properties were prepared by use of a facile method. These compounds were fully characterized by use of (1)H, (13)C, (31)P NMR, FT-IR, and elemental analyses. The thermal properties of these compounds including melting points and decomposition temperatures were investigated using DSC and TGA analyses. In addition, the paramagnetism, luminescence, and tumor targeting properties of these multifunctional compounds were confirmed by respective use of SQUID, fluorescence, and cell cytotoxicity studies. All compounds exhibited paramagnetism at room temperature, which could provide target delivery of these compounds to parts of the body containing tumor cells using a strong external magnetic field. In addition, these compounds display two major characteristic emissions originating from Dy(3+), which can be utilized for imaging tumor cells. The IC(50) values of these compounds measured against normal breast cell line (Hs578Bst) are significantly greater than those measured against the corresponding carcinoma breast cell line (Hs578T), clearly indicating the selective tumor targeting properties of these compounds. Confocal fluorescence microscopy studies were used to confirm the yellowish-green fluorescence corresponding to the emission of dysprosium thiocyanate anion within cancer cells upon exposure of cancer cell lines such as human pancreatic carcinoma cell line (MIAPaCa-2) and human breast carcinoma (MDA-MB-231) to a solution of these phosphonium-dysprosium compounds.

Laser capture microdissection of pancreatic ductal adeno-carcinoma cells to analyze EzH2 by Western Blot analysis.

Pure populations of tumor cells are essential for the identification of tumor-associated proteins for the development of targeted therapy. In recent years, laser capture microdissection (LCM) has been used successfully to obtain distinct populations of cells for subsequent molecular analysis. The polycomb group (PcG) protein, enhancer of zeste homolog 2 (EzH2), a methyl-transferase that plays a key role in -transcriptional gene repression, is frequently overexpressed in several malignant tumors. High levels of EzH2 are often associated with advanced disease stage in many solid tumors; however, its role in the pathogenesis of pancreatic ductal adeno-carcinoma (PDAC) is poorly understood. Because of the limited sample availability and the absence of in vitro amplification steps for proteins, the use of LCM for proteomics studies largely depends on highly sensitive protein detection methods. Here, we developed a faster and sensitive Western blot protocol and validated it for the detection of EzH2 in ∼2,000 cells. Initially, cultured PANC-1 cells were used to optimize protein electrophoresis and western blotting conditions. Gradient gel electrophoresis in combination with optimized antibody concentrations, and a sensitive chemiluminescent assay provided a strong signal. In order to further confirm the role of EzH2 in PDAC, employing siRNA-mediated gene silencing via long lasting plasmid vectors containing shRNA, we investigated the potential role of EzH2 gene silencing in pancreatic cancer regression. Positive correlation of EzH2 expression was observed with advanced stage, serous histology, and increasing grade in pancreatic cancer patient tissues. Further EzH2 knockdown resulted in decreased cell growth and invasiveness. The findings of this study emphasize that western blotting of a LCM-generated pure population of cancer cells may be a valuable technique for the study of tumor-specific proteins.

Peptide targeting of platinum anti-cancer drugs.

Besides various side effects caused by platinum anticancer drugs, they are not efficiently absorbed by the tumor cells. Two Pt-peptide conjugates; cyclic mPeg-CNGRC-Pt (7) and cyclic mPeg-CNGRC-Pten (8) bearing the Asn-Gly-Arg (NGR) targeting sequence, a malonoyl linker, and low molecular weight miniPEG groups have been synthesized. The platinum ligand was attached to the peptide via the carboxylic end of the malonate group at the end of the peptide. The pegylated peptide is nontoxic and highly soluble in water. Platinum conjugates synthesized using the pegylated peptides are also water-soluble with reduced or eliminated peptide immunogenicity. The choice of carboplatin as our untargeted platinum complex was due to the fact that the malonate linker chelates platinum in a manner similar to that of carboplatin. Cell toxicity assay and competition assay on the PC-3 cells (CD13 positive receptors) revealed selective delivery and destruction of PC-3 cells using targeted Pt-peptide conjugates 7 and 8 significantly more than untargeted carboplatin. Platinum uptake on PC-3 cells was 12-fold more for conjugate 7 and 3-fold more for conjugate 8 compared to that of the untargeted carboplatin, indicating selective activation of the CD13 receptors and delivery of the conjugates to CD13 positive cells. Further analysis on effects of conjugates 7 and 8 on PC-3 cells using caspase-3/7, fluorescence microscopy, and DNA fragmentation confirmed that the cells were dying by apoptosis.

Mouse apolipoprotein B editing complex 3 (APOBEC3) is expressed in germ cells and interacts with dead-end (DND1).

BACKGROUND: The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3'-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites. CONCLUSIONS/SIGNIFICANCE: The 3'-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.

Growth suppression of lung cancer cells by targeting cyclic AMP response element-binding protein.

Genes regulated by cyclic AMP-response element-binding protein (CREB) have been reported to suppress apoptosis, induce cell proliferation, and mediate inflammation and tumor metastasis. However, it is not clear whether CREB is critically involved in lung carcinogenesis. We found that non-small cell lung cancer (NSCLC) cell lines exhibited elevated constitutive activity in CREB, in its immediate upstream kinases (ribosomal s6 kinase and extracellular signal kinase), and in the CREB-regulated cell survival proteins Bcl-2 and Bcl-xL. We hypothesized that constitutively active CREB is important to lung cancer cell growth and survival and therefore could be a potential therapeutic target for NSCLC. Ectopic expression of dominant repressor CREB and transfection with small interfering RNA against CREB suppressed the growth and survival of NSCLC cells and induced apoptotic cell death. Furthermore, treating H1734 NSCLC cells with an inhibitor of the CREB signaling pathway Ro-31-8220 inhibited CREB activation by blocking the activity of extracellular signal kinase and ribosomal s6 kinase, arrested the cell cycle at the G(2)-M phase, and subsequently induced apoptosis with the suppression of Bcl-2 and Bcl-xL expression. Ro-31-8220 suppressed both the anchorage-dependent and independent growth of NSCLC cells, but its cytotoxic effect was much less prominent in normal bronchial epithelial cells. Our results indicate that active CREB plays an important role in NSCLC cell growth and survival. Thus, agents that suppress CREB activation could have potential therapeutic value for NSCLC treatment.

The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability.

Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain.

Nonclassical action of retinoic acid on the activation of the cAMP response element-binding protein in normal human bronchial epithelial cells.

Vitamin A (retinol) is essential for normal regulation of cell growth and differentiation. We have shown that the retinol metabolite retinoic acid (RA) induces mucous cell differentiation of normal human tracheobronchial epithelial (NHTBE) cells. However, early biological effects of RA in the differentiation of bronchial epithelia are largely unknown. Here, we showed that RA rapidly activated cAMP response element-binding protein (CREB). However, RA did not use the conventional retinoic acid receptor (RAR)/retinoid X receptor (RXR) to activate CREB. RA activated CREB in NHTBE and H1734 cells in which RARs/RXR were silenced with small interfering RNA (siRNA) targeting RAR/RXR expression or deactivated by antagonist. Inhibition of protein kinase C (PKC) or extracellular regulated kinase (ERK1/2) blocked the RA-mediated activation of CREB. In addition, depletion of p90 ribosomal S6 kinase (RSK) via siRSK1/2 completely abolished the activation, suggesting that PKC, ERK, and RSK are required for the activation. Altogether, this study provides the first evidence that RA rapidly activates CREB transcription factor via PKC, ERK, and RSK in a retinoid receptor-independent manner in normal bronchial epithelial cells. This noncanonical RA signaling pathway may play an important role in mediating early biological effects in the mucociliary differentiation of bronchial epithelia.

Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation.

Curcumin (diferuloylmethane), an anti-inflammatory agent used in traditional medicine, has been shown to suppress cellular transformation, proliferation, invasion, angiogenesis, and metastasis through a mechanism not fully understood. Because several genes that mediate these processes are regulated by nuclear factor-kappaB (NF-kappaB), we have postulated that curcumin mediates its activity by modulating NF-kappaB activation. Indeed, our laboratory has shown previously that curcumin can suppress NF-kappaB activation induced by a variety of agents (J Biol Chem 270:24995-50000, 1995). In the present study, we investigated the mechanism by which curcumin manifests its effect on NF-kappaB and NF-kappaB-regulated gene expression. Screening of 20 different analogs of curcumin showed that curcumin was the most potent analog in suppressing the tumor necrosis factor (TNF)-induced NF-kappaB activation. Curcumin inhibited TNF-induced NF-kappaB-dependent reporter gene expression in a dose-dependent manner. Curcumin also suppressed NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)1, TNFR2, NF-kappaB-inducing kinase, IkappaB kinase complex (IKK), and the p65 subunit of NF-kappaB. Such TNF-induced NF-kappaB-regulated gene products involved in cellular proliferation [cyclooxygenase-2 (COX-2), cyclin D1, and c-myc], antiapoptosis [inhibitor of apoptosis protein (IAP)1, IAP2, X-chromosome-linked IAP, Bcl-2, Bcl-x(L), Bfl-1/A1, TNF receptor-associated factor 1, and cellular Fas-associated death domain protein-like interleukin-1beta-converting enzyme inhibitory protein-like inhibitory protein], and metastasis (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1) were also down-regulated by curcumin. COX-2 promoter activity induced by TNF was abrogated by curcumin. We found that curcumin suppressed TNF-induced nuclear translocation of p65, which corresponded with the sequential suppression of IkappaBalpha kinase activity, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Curcumin also inhibited TNF-induced Akt activation and its association with IKK. Glutathione and dithiothreitol reversed the effect of curcumin on TNF-induced NF-kappaB activation. Overall, our results indicated that curcumin inhibits NF-kappaB activation and NF-kappaB-regulated gene expression through inhibition of IKK and Akt activation.

Melanoma differentiation-associated gene-7/IL-24 gene enhances NF-kappa B activation and suppresses apoptosis induced by TNF.

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-kappaB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-kappaB activation and on TNF-induced NF-kappaB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-kappaB. However, TNF-induced NF-kappaB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-kappaB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-kappaB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-kappaB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-kappaB activation. TNF-induced expression of the NF-kappaB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-kappaB by IkappaBalpha kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-kappaB, but potentiates TNF-induced NF-kappaB activation and NF-kappaB-regulated gene expression. Potentiation of NF-kappaB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.

Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-kappaB signaling.

Increased expression of proinflammatory and proangiogenic factors are associated with aggressive tumor growth and decreased survival of patients with head and neck squamous cell carcinoma (HNSCC). In as much as genes that are regulated by nuclear factor NF-kappaB suppress apoptosis, induce proliferation, and mediate inflammation, angiogenesis and tumor metastasis, agents that suppress NF-kappaB activation have potential as treatment for various cancers including HNSCC. We demonstrate that all HNSCC cell lines expressed constitutively active NF-kappaB and IkappaBalpha kinase (IKK), which is needed for NF-kappaB activation. Treatment of MDA 686LN cells with curcumin (diferuloylmethane), a pharmacologically safe chemopreventive agent, inhibited NF-kappaB activation through abrogation of IKK. As a result expression of various cell survival and cell proliferative genes including Bcl-2, cyclin D1, IL-6, COX-2 and MMP-9 was suppressed. This, in turn, inhibits proliferation of all HNSCC cell lines, arrests cell cycle in G1/S phase (MDA 686LN) and induces apoptosis as indicated by upstream and downstream caspase activation, PARP cleavage, annexin V staining in MDA 686LN cells. Suppression of NF-kappaB by cell-permeable p65-based peptide and NBD peptide also inhibited the proliferation and induced apoptosis in these cells. Our results indicate that curcumin is a potent inhibitor of cell proliferation and an inducer of apoptosis in HNSCC through suppression of IKK-mediated NF-kappaB activation and of NF-kappaB-regulated gene expression.