Evaluation of alkylating pyrrole-imidazole polyamide conjugates by a novel method for high-throughput sequencer

N-Methylpyrrole-N-methylimidazole (PI) polyamides are a class of DNA minor groove binders with DNA sequence-specificity. DNA-alkylating PI polyamide conjugates are attractive candidates as anticancer drugs acting through DNA damage and its subsequent inhibition of cell proliferation. One example is a chlorambucil-PI polyamide conjugate targeting the runt-related transcription factor (RUNX) family. RUNX1 has pro-oncogenic properties in acute myeloid leukemia, and recently the chlorambucil-PI polyamide conjugate was demonstrated to have anticancer effects. Herein, we apply another DNA-alkylating agent, seco-CBI, to target the consensus sequence of the RUNX family. Two types of CBI conjugates were prepared and their binding properties were characterized by Bind-n-Seq analysis using a high-throughput sequencer. The sequencing data were analyzed by two methods, MERMADE and our new MR (motif identification with a reference sequence), and the resultant binding motif logos were as predicted from the pairing rules proposed by Dervan et al. This is the first report to employ the MR method on alkylating PI polyamide conjugates. Moreover, cytotoxicity of conjugates 3 and 4 against a human non-small cell lung cancer, A549, were examined to show promising IC[50]s of 120 nm and 63 nm, respectively. These findings suggest seco-CBI-PI polyamide conjugates are candidates for oncological therapy

1,4,5,8-Tetra­isopropyl­anthracene

The mol­ecules of the title compound, C26H34, possess crystallographically imposed inversion symmetry. The anthracene ring system is planar within 0.038 (1) Å. The two methyl groups in each independent isopropyl group are oriented on either side of the anthracene plane. In the crystal structure, the mol­ecules adopt a herringbone-like arrangement without π–π stacking

1,7-Diethyl-4,10-diisopropyl­tetra­cene

The mol­ecule of the title compound, C28H32, is located on a crystallographic inversion center. The ethyl groups are essentially coplanar with the tetra­cene ring, making a torsion angle of −0.4 (4)°. The isopropyl groups adopt an asymmetric conformation with their terminal methyl groups positioned on opposite sides of the tetra­cene plane [the Me—C—C—C torsion angles are −22.5 (4) and 100.9 (3)°]. In the crystal, the mol­ecules adopt an arrangement without significant π–π inter­actions along the stacking direction (y axis)

1,4,5,8-Tetra-n-butyl­anthracene

The mol­ecule of the title compound, C30H42, occupies a special position on an inversion center. The four butyl side chains have all-trans planar conformations, and the alkyl planes are nearly orthogonal to the anthracene plane [C—C—C—C torsion angles of 79.6 (2) and 78.2 (2)°]. The overall mol­ecule has a stair-like shape with the n-butyl groups at the 1 and 8 positions extending towards the same side of the anthracene plane. In the crystal structure, mol­ecules adopt a slipped–parallel arrangement without π–π stacking

Activation of ASC induces apoptosis or necrosis, depending on the cell type, and causes tumor eradication

The adaptor protein ASC (also called TMS1) links certain NLR proteins (e.g., NLRC4, NLRP3) and caspases. It is involved in the chemosensitivity of tumor cells and inflammation. Here, we found that ASC activation using NLRC4 mimicry or an autoinflammatory disease-associated NLRP3 mutant induced necrosis in COLO205 colon adenocarcinoma cells, but induced caspase-8-dependent apoptosis in NUGC-4 stomach cancer cells. As the Fas ligand induced caspase-8-dependent apoptosis in COLO205 cells, caspase-8 was intact in this cell line. ASC-mediated necrosis was preceded by lysosomal leakage, and diminished by inhibitors for vacuolar H+-ATPase, cathepsins, and calpains but not by inhibitors for caspase-8, or aspartic proteases, suggesting that lysosomes and certain proteases were involved in this process. Finally, growing tumors of transplanted human cancer cells in nude mice were eradicated by the activation of endogenous ASC in the tumor cells, irrespective of the form of cell death. Thus, ASC mediates distinct forms of cell death in different cell types, and is a promising target for cancer therapy. (Cancer Sci 2010). © 2010 Japanese Cancer Association

Photodynamic Therapy With YAG-OPO Laser for Early Stage Lung Cancer

Photodynamic therapy (PDT) utilizing Photofrin is proving to be effective for the treatment of early stage lung cancers. The effect of PDT utilizing YAG-OPO laser as new light source was evaluated in 26 patients (29 lesions) with early stage lung cancers. YAG-OPO laser is solid state tunable laser which is easy to change wavelength between 620 and 670 nm exciting various kinds of photosensitizers. Moreover, YAG-OPO laser is more reliable, smaller and has less consumables than argon-dye laser or excimer-dye laser. As the result of PDT with YAG-OPO laser, complete remission (CR) was obtained in 82.6% of the 29 lesions, partial remission (PR) in 13.8% and no change (NC) was obtained in 3.4%. We conclude that PDT utilizing YAG-OPO laser is efficacious in the treatment of early stage lung cancers and can achieve complete remission

Mechanism of ASC-mediated apoptosis: Bid-dependent apoptosis in type II cells

金沢大学がん研究所がん病態制御Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor molecule that mediates apoptotic and inflammatory signals, and implicated in tumor suppression. However, the mechanism of ASC-mediated apoptosis has not been well elucidated. Here, we investigated the molecular mechanisms of ASC-mediated apoptosis in several cell lines using a caspase recruitment domain 12-Nod2 chimeric protein that transduces the signal from muramyl dipeptide into ASC-mediated apoptosis. Experiments using dominant-negative mutants, small-interfering RNAs and peptide inhibitors for caspases indicated that caspase-8 was generally required for ASC-mediated apoptosis, whereas a requirement for caspase-9 depended on the cell type. In addition, caspase-like apoptosis-regulatory protein (CLARP)/Fas-like inhibitor protein, a natural caspase-8 inhibitor, suppressed ASC-mediated apoptosis, and Clarp-/- mouse embryonic fibroblasts were highly sensitive to ASC-mediated apoptosis. Bax-deficient HCT116 cells were resistant to ASC-mediated apoptosis as reported previously, although we failed to observe colocalization of ASC and Bax in cells. Like Fas-ligand-induced apoptosis, the ASC-mediated apoptosis was inhibited by Bcl-2 and/or Bcl-XL in type-II but not type-I cell lines. Bid was cleaved upon ASC activation, and suppression of endogenous Bid expression using small-interfering RNAs in type-II cells reduced the ASC-mediated apoptosis. These results indicate that ASC, like death receptors, mediates two types of apoptosis depending on the cell type, in a manner involving caspase-8. © 2007 Nature Publishing Group All rights reserved

Latent trajectory modelling of pulmonary artery pressure in systemic sclerosis: a retrospective cohort study

OBJECTIVES: To visualise the trajectories of pulmonary arterial pressure (PAP) in systemic sclerosis (SSc) and identify the clinical phenotypes for each trajectory, by applying latent trajectory modelling for PAP repeatedly estimated by echocardiography. METHODS: This was a multicentre, retrospective cohort study conducted at four referral hospitals in Kyoto, Japan. Patients with SSc who were treated at study sites between 2008 and 2021 and who had at least three echocardiographic measurements of systolic PAP (sPAP) were included. A group-based trajectory model was applied to the change in sPAP over time, and patients were classified into distinct subgroups that followed similar trajectories. Pulmonary hypertension (PH)-free survival was compared for each trajectory. Multinomial logistic regression analysis was performed for baseline clinical characteristics associated with trajectory assignment. RESULTS: A total of 236 patients with 1097 sPAP measurements were included. We identified five trajectories: rapid progression (n=9, 3.8%), early elevation (n=30, 12.7%), middle elevation (n=54, 22.9%), late elevation (n=24, 10.2%) and low stable (n=119, 50.4%). The trajectories, in the listed order, showed progressively earlier elevation of sPAP and shorter PH-free survival. In the multinomial logistic regression analysis with the low stable as a reference, cardiac involvement was associated with rapid progression, diffuse cutaneous SSc was associated with early elevation and anti-centromere antibody was associated with middle elevation; older age of onset was associated with all three of these trajectories. CONCLUSION: The pattern of changes in PAP over time in SSc can be classified into five trajectories with distinctly different clinical characteristics and outcomes

IL-10 Inhibits Transforming Growth Factor-ß-Induction of Type I Collagen mRNA Expression via Both JNK and p38 Pathways in Human Lung Fibroblasts

Transforming growth factor-ß (TGF-ß) is a key factor for understanding the pathogenesis of fibrotic disorders such as idiopathic pulmonary fibrosis (IPF). We have demonstrated that interleukin-10 (IL-10) suppresses TGF-ß-induced expression of type I collagen (COL1) mRNA in a human lung fibroblast cell line (WI-38). However, the inhibitory mechanism has not yet been clearly elucidated. Thus, in the current study, we investigate the effects of IL-10 blockade of TGF-ß signaling which regulates COL1 mRNA expression. In WI-38 cells, IL-10 inhibits TGF-ß-mediated phosphorylation of both, c-Jun HN2-terminal kinase (JNK) and p38, but does not suppress TGF-ß- mediated phosphorylation of Smad2 or affect TGF-ß-upregulation of Smad7 mRNA expression. In addition, SP600125 and SB203580, specific inhibitors of JNK and p38, respectively, attenuate TGF-ß-induced COL1 mRNA expression in WI-38 cells. These results suggest that IL-10 inhibits TGF-ß-induced COL1 mRNA expression via both JNK and p38 pathways but not Smad pathways in WI-38 cells. This inhibitory mechanism may provide a novel insight into therapeutic strategies for fibrotic disorders such as IPF