Stereospecific inhibition of AMPK by (R)-crizotinib induced changes to the morphology and properties of cancer and cancer stem cell-like cells.

Crizotinib is used in the clinic for treating patients with ALK- or ROS1-positive non-small-cell lung carcinoma. The objective of the present study was to determine if crizotinib enantiomers could induce changes to the properties of cancer and cancer stem cell (CSC)-like cells at a high concentration (∼ 3 µM). While (R)-crizotinib induced changes in morphologies or sizes of cells, (S)-crizotinib did not. Pretreatment with (R)-crizotinib suppressed the proliferation of cancer or CSC-like cells in vitro and tumor growth in vivo. In vivo administration of (R)-crizotinib inhibited the growth of tumors formed from CSC-like cells by 72%. %. Along with the morphological changes induced by (R)-crizotinib, the expression levels of CD44 (NCI-H23 and HCT-15), ALDH1 (NCI-H460), nanog (PC-3), and Oct-4A (CSC-like cells), which appear to be specific marker proteins, were greatly changed, suggesting that changes in cellular properties accompanied the morphological changes in the cells. The expression levels of Snail, Slug, and E-cadherin were also greatly altered by (R)-crizotinib. Among several signal transduction molecules examined, AMPK phosphorylation appeared to be selectively inhibited by (R)-crizotinib. BML-275 (an AMPK inhibitor) and AMPKα2 siRNA efficiently induced morphological changes to all types of cells examined, suggesting that (R)-crizotinib might cause losses of characteristics of cancer or CSCs via inhibition of AMPK. These results indicate that (R)-crizotinib might be an effective anticancer agent that can cause alteration in cancer cell properties.

Cryogenic ion spectroscopy of adenine complexes containing alkali metal cations.

Cryogenic ion spectroscopy was used to characterize adenine complexes containing alkali metal cations (M+A, M = Cs, Rb, K, Na, and Li) produced by electrospray ionization. The ultraviolet (UV) photodissociation spectra of the complexes stored in a cryogenic ion trap exhibited well-resolved vibronic bands near their origin bands of the S0-S1 transition. The UV-UV hole-burning and infrared ion-dip spectra showed that all the M+A ions in the ion trap were single isomers of M+A7a, where the M+ ion was not bound to canonical 9H-adenine (A9) but bound to a rare tautomer, 7H-adenine (A7). Density functional theory calculations showed lower tautomerization barriers for M+A9 than for bare A9 in aqueous solution. We suggest that M+ ions not only play a catalytic role in the tautomerization of A9 to A7 but also increase the tautomerization yield by forming stable M+A7a isomers.

Author Correction: Structure-Activity Relationships of Baicalein and its Analogs as Novel TSLP Inhibitors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure-Activity Relationships of Baicalein and its Analogs as Novel TSLP Inhibitors.

Thymic stromal lymphopoietin (TSLP) plays an important role in the differentiation and proliferation of Th2 cells, resulting in eosinophilic inflammation and numerous allergic diseases. Baicalein (1), a major component of Scutellaria baicalensis, was found to be the first small molecule to block TSLP signaling pathways. It inhibited effectively eosinophil infiltration in house dust mite-induced and ovalbumin-challenged mouse models. Structure-activity relationship studies identified compound 11a, a biphenyl flavanone analog, as a novel human TSLP inhibitor for the discovery and development of new anti-allergic drugs.

Dopamine receptor antagonists induce differentiation of PC-3 human prostate cancer cell-derived cancer stem cell-like cells.

BACKGROUND: The objective of this study was to determine whether PC-3 human prostate cancer cell-derived cancer stem cells (CSC)-like cells grown in a regular cell culture plate not coated with a matrix molecule might be useful for finding differentiation-inducing agents that could alter properties of prostate CSC. METHODS: Monolayer cells prepared from sphere culture of PC-3 cells were characterized for the presence of pluripotency and tumorigenicity. They were then applied to screen a compound library to find compounds that could induce morphology changes of cells. Mechanisms of action of compounds selected from the chemical library that induced the loss of pluripotency of cells were also investigated. RESULTS: C5A cells prepared from PC-3 cell-derived sphere culture expressed pluripotency markers such as Oct4, Sox2, and Klf4. C5A cells were highly proliferative. They were invasive in vitro and tumorigenic in vivo. Some dopamine receptor antagonists such as thioridazine caused reduction of pluripotency markers and tumorigenicity. Thioridazine, unlike promazine, inhibited phosphorylation of AMPK in a dose dependent manner. BML-275, an AMPK inhibitor, also induced differentiation of C5A cells as seen with thioridazine whereas A769663, an AMPK activator, blocked its differentiation-inducing ability. Transfection of C5A cells with siRNAs of dopamine receptor subtypes revealed that knockdown of DRD2 or DRD4 induced morphology changes of C5A cells. CONCLUSIONS: Some dopamine receptor antagonists such as thioridazine can induce differentiation of CSC-like cells by inhibiting phosphorylation of AMPK. Binding to DRD2 or DRD4 might have mediated the action of thioridazine involved in the differentiation of CSC-like cells.

Monoclonal antibodies against autocrine motility factor suppress gastric cancer.

Autocrine motility factor (AMF), which is a secreted form of phosphoglucose isomerase, is mainly secreted by various tumors and has cytokine-like activity. AMF is known to stimulate proliferation, survival and metastasis of cancer cells, and angiogenesis within a tumor. The present study investigated whether inhibition of AMF using targeted-antibodies was able to suppress the growth of cancer. A migration assay using a Boyden chamber was utilized to measure the activity of AMF on the motility of cancer cells. A recombinant human AMF (rhAMF) prepared from E. coli transformed with the pET22b-AMF vector increased the motility of MDA-MB-231 and A549 cells, but it did not affect that of NCI-N87 or HepG2 cells, which exhibited the ability to secrete high amounts of their own endogenous AMF into the culture medium. The extent to which the AMF receptor was expressed on cancer cells did not correlate clearly with the cell motility stimulated by rhAMF. In A549-xenografted nude mice treated with sunitinib or cetuximab, a decrease in the plasma AMF concentration was accompanied by a reduction in tumor weight, suggesting an association between the plasma AMF concentration and anticancer activity. A monoclonal antibody (9A-4H), which revealed a high binding affinity for E. coli-derived rhAMF, significantly suppressed the growth of tumors in Balb/c nude mice transplanted with the human gastric cancer cell line NCI-N87, to the similar extent as trastuzumab, an anticancer antibody. The present study suggests, for the first time, that an antibody specific to AMF may be a therapeutic agent for gastric cancer.

Breast cancer cells evade paclitaxel-induced cell death by developing resistance to dasatinib.

Triple negative breast cancer (TNBC), which does not express the progesterone, estrogen, or HER2/neu receptor, is aggressive and difficult to treat. Paclitaxel, a tubulin stabilizing agent, is one of the most frequently prescribed anticancer agents for breast cancers, including TNBC. Residual disease that occurs due to resistance or partial resistance of cancer cells in a tumor against anticancer agents is the most important issue in oncology. In the present study, when MDA-MB-231 cells, a TNBC cell line, were treated with 30 µM paclitaxel, a slightly higher concentration than its GI50 value, for 6 days, a small number of cells with different morphologies survived. Among the surviving cells, small round cells were isolated, cloned, and named MDA-MB-231-JYJ cells. MDA-MB-231-JYJ cells were observed to be highly proliferative and tumorigenic. In addition, signal transduction molecules involved in proliferation, survival, malignancy, or stemness of cancer cells, such as c-Src, c-Met, Notch 1, c-Myc, Sox2, Oct3/4, Nanog, and E-cadherin were highly expressed or activated. While further study is required, MDA-MB-231-JYJ cells appear to have some of the characteristics of cancer precursor cells. Although MDA-MB-231-JYJ cells were isolated from the cells that survived in the continuous presence of paclitaxel, they were not resistant to paclitaxel but developed resistance to dasatinib, a Bcr-Abl and Src kinase family inhibitor. The activated state of Src and Notch 1, and the expression levels of c-Myc and cyclins in MDA-MB-231-JYJ cells were less affected than MDA-MB-231 cells by the treatment of dasatinib, which may explain the resistance of MDA-MB-231-JYJ cells to dasatinib. These results suggest that cancer cells that become resistant to dasatinib during the process of paclitaxel therapy in patients may appear, and caution is required in the design of clinical trials using these two agents.

NgR1 Expressed in P19 Embryonal Carcinoma Cells Differentiated by Retinoic Acid Can Activate STAT3.

NgR1, a Nogo receptor, is involved in inhibition of neurite outgrowth and axonal regeneration and regulation of synaptic plasticity. P19 embryonal carcinoma cells were induced to differentiate into neuron-like cells using all trans-retinoic acid and the presence and/or function of cellular molecules, such as NgR1, NMDA receptors and STAT3, were examined. Neuronally differentiated P19 cells expressed the mRNA and protein of NgR1, which could stimulate the phosphorylation of STAT3 when activated by Nogo-P4 peptide, an active segment of Nogo-66. During the whole period of differentiation, mRNAs of all of the NMDA receptor subtypes tested (NR1, NR2A-2D) were consistently expressed, which meant that neuronally differentiated P19 cells maintained some characteristics of neurons, especially central nervous system neurons. Our results suggests that neuronally differentiated P19 cells expressing NgR1 may be an efficient and convenient in vitro model for studying the molecular mechanism of cellular events that involve NgR1 and its binding partners, and for screening compounds that activate or inhibit NgR1.

Involvement of human histamine N-methyltransferase gene polymorphisms in susceptibility to atopic dermatitis in korean children.

PURPOSE: Histamine N-methyltransferase (HNMT) catalyzes one of two major histamine metabolic pathways. Histamine is a mediator of pruritus in atopic dermatitis (AD). The aim of this study was to evaluate the association between HNMT polymorphisms and AD in children. METHODS: We genotyped 763 Korean children for allelic determinants at four polymorphic sites in the HNMT gene: -465T>C, -413C>T, 314C>T, and 939A>G. Genotyping was performed using a TaqMan fluorogenic 5' nuclease assay. The functional effect of the 939A>G polymorphism was analyzed. RESULTS: Of the 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of the HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P=0.004), and those of HNMT 939A>G were significantly associated with eczema in the atopy groups (P=0.048). Frequency distributions of HNMT -465T>C and -413C>T were not associated with eczema. Subjects who were AA homozygous or AG heterozygous for 939A>G showed significantly higher immunoglobulin E levels than subjects who were GG homozygous (P=0.009). In U937 cells, the variant genotype reporter construct had significantly higher mRNA stability (P<0.001) and HNMT enzyme activity (P<0.001) than the common genotype. CONCLUSIONS: Polymorphisms in HNMT appear to confer susceptibility to AD in Korean children.

Alpha-eleostearic acid induces autophagy-dependent cell death through targeting AKT/mTOR and ERK1/2 signal together with the generation of reactive oxygen species.

Alpha-eleostearic acid (alpha-ESA, 9Z11E13E-18:3), a linolenic acid isomer with a conjugated triene system, is a natural and biologically-active compound that has been shown to possess potent anti-tumor properties. Herein, we demonstrate alpha-ESA induced apoptosis and autophagy with reactive oxygen species (ROS) generation in HeLa cells. Treatment with alpha-ESA caused inhibition of phosphorylated (p)AKT and elongated the sub G1 phase in the cell cycle, indicating induction of apoptosis. Autophagy was also induced by alpha-ESA treatment, causing low pAKT and pP70S6K activities, increasing pERK1/2 and leading to a higher conversion rate of LC3 I to LC3 II compared to that of the control. The autophagy was further confirmed by fluorescence microscopy and flow cytometry through monodansylcadavarine (MDC) staining. It appears that the role of autophagy is a protective mechanism against cell death in alpha-ESA-treated HeLa cells. Subsequently, we found that treating HeLa cells with alpha-ESA induced the generation of reactive oxygen species (ROS). The phosphorylation of P70S6K, downstream of mTOR signaling, and AKT were further reduced by pretreatment with N-acetyl-l-cysteine (NAC), an ROS scavenger, whereas the phosphorylation of ERK1/2 and the conversion of LC3 I to LC3 II were further enhanced. As a result, the blocking of the action of ROS promoted alpha-ESA-induced apoptosis and autophagy. Taken together, our results indicate that the generation of ROS by alpha-ESA treatment impedes the progress of apoptosis and excessive autophagy formation which takes part in cell death, thus impeding death promotion.