Novel non-trimethoxylphenyl piperlongumine derivatives selectively kill cancer cells

Piperlongumine (PL) is a natural alkaloid with broad biological activities. Twelve analogues have been designed and synthesized with non-substituted benzyl rings or heterocycles in this work. Most of the compounds showed better anticancer activities than the parent PL without apparent toxicity in normal cells. Elevation of cellular ROS levels was one of the main anticancer mechanisms of these compounds. Cell apoptosis and cell cycle arrest for the best compound ZM90 were evaluated and similar mechanism of action with PL was demonstrated. The SAR was also characterized, providing worthy directions for further optimization of PL compounds.     

Phototoxic aptamers selectively enter and kill epithelial cancer cells

The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e₆ and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells.     

DU-145 prostate carcinoma cells that selectively transmigrate narrow obstacles express elevated levels of Cx43

The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the “leading front” formation during cancer invasion.     

TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Here we identified T cell immunoglobulin mucin-3 (TIM-3) as a surface molecule expressed on LSCs in most types of AML except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). TIM-3(+) but not TIM-3? AML cells reconstituted human AML in immunodeficient mice, suggesting that the TIM-3(+) population contains most, if not all, of functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden and eliminated LSCs capable of reconstituting human AML in secondary recipients. These data suggest that TIM-3 is one of the promising targets to eradicate AML LSCs.     

Comparison of ras-responsive gene enhancers in pancreatic tumor cells that express either wild-type or mutant K-ras

There is a pressing need for new therapies to treat pancreatic cancer. In principle, this could be achieved by taking advantage of signaling pathways that are active in tumor, but not normal, cells. The work described in this study set out to determine whether the activities of three enhancers, which have been reported to be highly responsive to activated ras, differ in pancreatic tumor cells that express wild-type versus constitutively active mutant forms of K-ras. Surprisingly, the three enhancers are active in four different pancreatic tumor cell lines that express either normal K-ras gene or mutant K-ras. Moreover, reducing the concentration of serum in the growth medium from 10% to 0.5% had relatively little effect on the strength of any of the enhancers, although it drastically affected cell growth. Importantly, our studies also indicate that MEK is active in pancreatic tumor cells that possess wild-type as well as mutant K-ras, even when cultured in medium that severely limits cell growth. These findings support the hypothesis that the Ras/Raf/Mek/Erk pathway may be constitutively active even in pancreatic tumor cells that express wild-type K-ras.     

Painful research: identification of a small-molecule inhibitor that selectively targets Nav1.8 sodium channels

Voltage-gated sodium channels in nociceptive neurons are attractive targets for novel pain therapeutics. Although drugs that target voltage-gated sodium channels have proven value as pain therapeutics, the drugs that are currently available are non-specific sodium channel inhibitors, which limit their usefulness. Recently, a selective small-molecule inhibitor of Na(v)1.8, a voltage-gated sodium channel isoform that participates in peripheral pain mechanisms, has been developed. This exciting new compound shows efficacy in several animal models of pain and is anticipated to be only the first of many new isoform-specific sodium channel blockers.     

Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac alpha-myosin transgene including the R(403)Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the beta-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled beta-adrenergic receptor kinase 1 (beta-ARK1) were increased by more than twofold over controls, and total beta-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.     

Development of novel antibacterial peptides that kill resistant isolates

The rapid emergence of bacterial strains that are resistant to current antibiotics requires the development of novel types of antimicrobial compounds. Proline-rich cationic antibacterial peptides such as pyrrhocoricin kill responsive bacteria by binding to the 70 kDa heat shock protein DnaK and inhibiting protein folding. We designed and synthesized multiply protected dimeric analogs of pyrrhocoricin and optimized the in vitro antibacterial efficacy assays for peptide antibiotics. Pyrrhocoricin and the designed dimers killed β-lactam, tetracycline- or aminoglycoside-resistant strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the submicromolar or low micromolar concentration range. One of the peptides also killed Pseudomonas aeruginosa. The designed dimers showed improved stability in mammalian sera compared to the native analog. In a murine H. influenzae lung infection model, a single dose of a dimeric pyrrhocoricin analog reduced the bacteria in the bronchoalveolar lavage when delivered intranasally. The solid-phase synthesis was optimized for large-scale laboratory preparations.     

Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage

Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.     

Possible role of natural killer cells in negative selection of mutant lymphocytes that fail to express the human leukocyte antigen-A2 allele

Increased frequencies of cells carrying mutations at several loci have been found in the blood cells of atomic-bomb (A-bomb) survivors upon testing four or five decades after the bombing. Interestingly, though, we have been unable to demonstrate any radiation-associated increases in the frequencies of mutant blood cells in which human leukocyte antigen (HLA)-A expression has been disrupted; this is true both of preliminary tests on the T cells of a small subset of A-bomb survivors and of the much more extensive study reported here in which we screened a much larger group of survivors for HLA-A2 loss mutations in B cells and granulocytes as well as in T cells. In attempting to explain our inability to detect any increases in HLA-A2-negative cell numbers in HLA-A2 heterozygous individuals exposed to A-bomb irradiation, we decided to test the hypothesis that HLA-A mutant lymphocytes might well have been induced by radiation exposure in much the same way as every other type of mutant we encountered, but may subsequently have been eliminated by the strong negative selection associated with their almost inevitable exposure to autologous natural killer (NK) cells in the bloodstream of each of the individuals concerned. We now report that mutant B lymphocyte cell lines that have lost the ability to express the HLA-A2 antigen do indeed appear to be much more readily eliminated than their parental heterozygous counterparts during co-culture in vitro with autologous NK cells. We make this claim first because we have observed that adding autologous NK cells to in vitro cultures of HLA-A2 heterozygous B or T cell lines appeared to cause a dose-dependent decrease in the numbers of HLA-A2-negative mutants that could be detected over a period of 3 days, and second because when we used peripheral blood HLA-A2 heterozygous lymphocyte cultures from which most of the autologous NK cells had been removed we found that we were able to detect newly-arising HLA-A2 mutant T cells in substantial numbers. Taken together, these results strongly support the hypothesis that autologous NK cells are responsible for eliminating mutant lymphocytes that have lost the ability to express self-HLA class I molecules in vivo, and may well therefore explain why we have been unable to detect increased frequencies of HLA-A2 mutants in samples from any of the 164 A-bomb survivors whose HLA-A2 heterozygote status made their lymphocytes suitable for our tests.     

Brown bears selectively kill salmon with higher energy content but only in habitats that facilitate choice

Pacific salmon return to spawn in thousands of streams across the Pacific Rim, from large rivers to tiny headwater streams. Once on the spawning grounds, salmon undergo dramatic biochemical changes as they metabolize stored lipid and protein reserves; at stream entrance, they will contain up to 85% more lipid and 40% more protein than at their senescent death a week or two later. Foraging brown and black bears that congregate at spawning streams thus encounter salmon that vary dramatically in their energy content and thus energetic reward. We hypothesized that bears would selectively kill salmon that are highest in energy content (fewest number of days on the spawning grounds) when they pursue salmon at small shallow streams where little effort is necessary to capture salmon, i.e. habitats that facilitate choice. In contrast, bears in environments where foraging is difficult (deeper, more complex streams) should be less selective and should capture salmon that are most available. We tested these ideas by examining predation rates on fish of different in-stream ages (i.e. energy content) at three different streams that varied in physical habitat attributes. At a very shallow, simple stream, bears preferentially killed salmon that had spent the fewest days in the stream. At two streams where deeper water and woody debris provided refuges for salmon, predation rates increased with in-stream age. At the shallowest streams encounter rates and capture success are likely equal among the high- and low-energy salmon and thus predation rates reflect active choice by bears. In contrast capture success probably increases on the older salmon at the larger streams (due to a loss of vigor), and thus 'preference' for these fish increases due to decreasing effort necessary to capture them.     

Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.     

Identification and characterization of a cell-surface molecule that is selectively induced on rat lymphokine-activated killer cells

We have identified a 40- to 45-kDa cell-surface molecule designated gp42, that is expressed in high levels by rat lymphokine-activated killer (LAK) cells of NK cell origin. gp42 cannot be detected on the precursors of LAK cells and is not present on resting or activated T cells. Rather, expression of gp42 is selectively induced on NK cells by the high concentrations of rIL-2 that are required for the induction of LAK activity. Although the function of gp42 is not known, the selective nature of its expression suggests a role for this molecule in regulating responses that are unique to IL-2-activated NK cells.     

Memory CD4 T cells that express CXCR5 provide accelerated help to B cells

CD4 T cell help for B cells is critical for effective Ab responses. Although many of the molecules involved in helper functions of naive CD4 T cells have been characterized, much less is known about the helper capabilities of memory CD4 T cells, an important consideration for the design of vaccines that aim to prime protective memory CD4 T cells. In this study, we demonstrate that memory CD4 T cells enable B cells to expand more rapidly and class switch earlier than do primary responding CD4 T cells. This accelerated response does not require large numbers of memory cells, and similar numbers of primary responding cells provide less effective help than do memory cells. However, only memory CD4 T cells that express the B cell follicle homing molecule, CXCR5, are able to accelerate the response, suggesting that the rapidity of the Ab response depends on the ability of CD4 memory T cells to migrate quickly toward B cells.     

FRET-based screening assay using small-molecule photoluminescent probes in lysate of cells overexpressing RFP-fused protein kinases

An assay was developed for the characterization of protein kinase inhibitors in lysates of mammalian cells based on the measurement of FRET between overexpressed red fluorescent protein (TagRFP)-fused protein kinases (PKs) and luminophore-labeled small-molecule inhibitors (ARC-Photo probes). Two types of the assay, one using TagRFP as the photoluminescence donor together with ARC-Photo probes containing a red fluorophore dye as acceptor, and the other using TagRFP as the acceptor fluorophore in combination with a terbium cryptate-based long-lifetime photoluminescence donor, were used for FRET-based measurements in lysates of the cells overexpressing TagRFP-fused PKs. The second variant of the assay enabled the performance of the measurements under time-resolved conditions that led to substantially higher values of the signal/background ratio and further improved the reliability of the assay.     

Human endothelial cells selectively express large amounts of pancreatic-type ribonuclease (RNase 1)

Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.     

Phenotype of a herpes simplex virus type 1 mutant that fails to express immediate-early regulatory protein ICP0

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) regulatory protein ICP0 is required for efficient progression of infected cells into productive lytic infection, especially in low-multiplicity infections of limited-passage human fibroblasts. We have used single-cell-based assays that allow detailed analysis of the ICP0-null phenotype in low-multiplicity infections of restrictive cell types. The major conclusions are as follows: (i) there is a threshold input multiplicity above which the mutant virus replicates normally; (ii) individual cells infected below the threshold multiplicity have a high probability of establishing a nonproductive infection; (iii) such nonproductively infected cells have a high probability of expressing IE products at 6 h postinfection; (iv) even at 24 h postinfection, IE protein-positive nonproductively infected human fibroblast cells exceed the number of cells that lead to plaque formation by up to 2 orders of magnitude; (v) expression of individual IE proteins in a proportion of the nonproductively infected cells is incompletely coordinated; (vi) the nonproductive cells can also express early gene products at low frequencies and in a stochastic manner; and (vii) significant numbers of human fibroblast cells infected at low multiplicity by an ICP0-deficient virus are lost through cell death. We propose that in the absence of ICP0 expression, HSV-1 infected human fibroblasts can undergo a great variety of fates, including quiescence, stalled infection at a variety of different stages, cell death, and, for a minor population, initiation of formation of a plaque.