Efficient entry into S phase of the cell cycle is necessary for embryonic development and tissue homoeostasis. However, unscheduled S phase entry triggers DNA damage and promotes oncogenesis, ...underlining the requirement for strict control. Here, we identify the NUCKS1-SKP2-p21/p27 axis as a checkpoint pathway for the G1/S transition. In response to mitogenic stimulation, NUCKS1, a transcription factor, is recruited to chromatin to activate expression of SKP2, the F-box component of the SCF
ubiquitin ligase, leading to degradation of p21 and p27 and promoting progression into S phase. In contrast, DNA damage induces p53-dependent transcriptional repression of NUCKS1, leading to SKP2 downregulation, p21/p27 upregulation, and cell cycle arrest. We propose that the NUCKS1-SKP2-p21/p27 axis integrates mitogenic and DNA damage signalling to control S phase entry. The Cancer Genome Atlas (TCGA) data reveal that this mechanism is hijacked in many cancers, potentially allowing cancer cells to sustain uncontrolled proliferation.
The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) ...has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.
Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling ...pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling) and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug) upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer.
Treatment of psoriatic arthritis with the oral Janus kinase inhibitor tofacitinib for 3 months was more effective than placebo in reducing joint manifestations, as determined by the American College ...of Rheumatology 20% response rate, but was associated with herpes infections.
1 Winters Center for Heart Failure Research, Michael E. DeBakey Department of Veterans Affairs Medical Center, and 3 Thrombosis Section, Baylor College of Medicine and Methodist Hospital, Houston, ...Texas; 2 Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom; and 4 Molecular Pharmacology Department, AstraZeneca R&D Molndal, AstraZeneca Pharmaceuticals, Gothenburg, Sweden
Submitted 29 April 2004
; accepted in final form 11 August 2004
The balance between matrix metalloproteinases (MMPs) and their natural inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), plays a critical role in cardiac remodeling. Although a number of studies have characterized the pathophysiological role of MMPs in the heart, very little is known with respect to the role of TIMPs in the heart. To delineate the role of TIMPs in the heart we examined the effects of adenovirus-mediated overexpression of TIMP-1, -2, -3, and -4 in cardiac fibroblasts. Infection of cardiac fibroblasts with adenoviral constructs containing human recombinant TIMP (AdTIMP-1, -2, -3, and -4) provoked a significant ( P < 0.0001) 1.3-fold in increase in bromodeoxyuridine (BrdU) incorporation. Similarly, treatment of cardiac fibroblasts with AdTIMP-1-, -2-, -3-, and -4-conditioned medium led to a 1.2-fold increase in BrdU incorporation ( P < 0.0001) that was abolished by pretreatment with anti-TIMP-1, -2, -3, and -4 antibodies. The effects of TIMPs were not mimicked by treating the cells with RS-130830, a broad-based MMP inhibitor, suggesting that the effects of TIMPs were independent of their ability to inhibit MMPs. Infection with AdTIMP-1, -2, -3, and -4 led to a significant increase in -smooth muscle actin staining, consistent with TIMP-induced phenotypic differentiation into myofibroblasts. Finally, infection with AdTIMP-2 resulted in a significant increase in collagen synthesis, whereas infection with AdTIMP-3 resulted in a significant increase in fibroblast apoptosis. TIMPs exert overlapping as well as diverse effects on isolated cardiac fibroblasts. The observation that TIMPs stimulate fibroblast proliferation as well as phenotypic differentiation into myofibroblasts suggests that TIMPs may play an important role in tissue repair in the heart that extends beyond their traditional role as MMP inhibitors.
matrix metalloproteinase; proliferation; apoptosis; phenotypic differentiation
Address for reprint requests and other correspondence: D. L. Mann, Winters Center for Heart Failure Research, MS 524, 6565 Fannin, Houston, TX 77030 (E-mail: dmann{at}bcm.tmc.edu )
Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman–Birk ...inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.
Mechanisms underlying the generation of induced pluripotent stem cells (iPSC) and keeping iPSC stability remain to be further defined. Accumulated evidences showed that iPSC reprogramming may be ...controlled by the cell-division-rate-dependent model. Here we reported effects of absence of mouse p27 or p18 on iPSC generation efficiency and genomic stability. Expression levels of cyclin-dependent kinases inhibitors (CDKIs), p21, p27, and p18 decreased during iPSC reprogramming. Like p21 loss, p27 or p18 deficiency significantly promoted efficiency of iPSC generation, whereas ectopic expression of p27, p18, or treatment with CDK2 or CDK4 inhibitors repressed the reprogramming rate, suggesting that CDKIs-regulated iPSC reprogramming is directly related with their functions as CDK inhibitors. However, unlike p21 deletion, absence of p27 or p18 did not increase DNA damage or chromosomal aberrations during iPSC reprogramming and at iPSC stage. Our data not only support that cell cycle regulation is critical for iPSC reprogramming, but also reveal the distinction of CDKIs in somatic cell reprogramming.
The actions of many drugs involve enzyme inhibition. This is exemplified by the inhibitors of monoamine oxidases (MAO) and the cholinsterases (ChE) that have been used for several pharmacological ...purposes. This review describes key principles and approaches for the reliable determination of enzyme activities and inhibition as well as some of the methods that are in current use for such studies with these two enzymes. Their applicability and potential pitfalls arising from their inappropriate use are discussed. Since inhibitor potency is frequently assessed in terms of the quantity necessary to give 50% inhibition (the IC
value), the relationships between this and the mode of inhibition is also considered, in terms of the misleading information that it may provide. Incorporation of more than one functionality into the same molecule to give a multi-target-directed ligands (MTDLs) requires careful assessment to ensure that the specific target effects are not significantly altered and that the kinetic behavior remains as favourable with the MTDL as it does with the individual components. Such factors will be considered in terms of recently developed MTDLs that combine MAO and ChE inhibitory functions.
Hypertension is accompanied by structural remodelling of vascular extracellular matrix (ECM). Tissue inhibitor of metalloproteinases (TIMPs) inhibits matrix metalloproteinases (MMPs) that degrade the ...matrix structural proteins. In response to a hypertensive stimulus, the balance between MMPs and TIMPs is altered. We examined the role of TIMPs in agonist-induced hypertension.
We subjected TIMP-knockout mice to angiotensin II (Ang II) infusion, and found that Ang-II-induced hypertension in TIMP1(-/-), TIMP2(-/-), and TIMP4(-/-) mice was comparable to wild-type (WT) mice, but significantly suppressed in TIMP3(-/-) mice. Ex vivo pressure myography analyses on carotid and mesenteric arteries revealed that Ang-II-infused TIMP3(-/-) arteries were more distensible with impaired elastic recoil compared with the WT group. The acute response to vasoconstriction and vasodilation was intact in TIMP3(-/-) mesenteric and carotid arteries. Mesenteric arteries from TIMP3(-/-)-Ang II mice exhibited a reduced media-to-lumen ratio, suppressed collagen and elastin levels, elevated elastase and gelatinase proteolytic activities compared with WT-Ang II. TIMP3(-/-)-Ang II carotid arteries also showed adverse structural remodelling. Treatment of mice with doxycycline, a matrix metalloproteinase inhibitor, improved matrix integrity in mesenteric and carotid arteries in TIMP3(-/-)-Ang II and differentially regulated elastin and collagen levels in WT-Ang II vs. TIMP3(-/-)-Ang II.
Our study demonstrates a critical role for TIMP3, among all TIMPs, is preserving arterial ECM in response to Ang II. It is critical to acknowledge that the suppressed Ang-II-induced hypertension in TIMP3(-/-) mice is not a protective mechanism but owing to adverse remodelling in arterial matrix.
Abstract Background and objectives S49076 is a novel ATP-competitive tyrosine kinase inhibitor of MET, AXL and FGFR with a unique selectivity profile. A phase I open-label study was undertaken to ...establish the tolerability profile and determine the recommended dose (RD) and administration schedule. Materials and methods Patients with advanced solid tumours received S49076 orally once-daily ( qd ) or twice-daily ( bid ) in continuous 21-day cycles at escalating doses guided by a 3 + 3 design and followed by an expansion phase at the RD. Pharmacokinetic (PK) parameters were assessed and pharmacodynamic end-points were evaluated in pre- and post-treatment tumour biopsies. Preliminary anti-tumour activity was evaluated as per the Response Evaluation Criteria In Solid Tumours 1.1 criteria. Results A total of 103 patients were treated: 79 in the dose-escalation and 24 in the expansion. Doses from 15 to 900 mg were evaluated. Dose-limiting toxicities were reported in 9 patients and occurred at 30, 760 and 900 mg in the qd arm and at 180, 225 and 285 mg in the bid arm. The RD was defined at 600 mg qd . Adverse events (AEs) occurred with similar frequency in both regimens at an equivalent total daily dose. Overall, 83 patients (81.4%) had drug-related AEs, the majority (93%) of which were grade I–II (National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0) and only 3% led to drug discontinuation. Intratumoural PK analysis at the RD suggested hitting of MET, AXL and FGFR. Conclusion S49076 demonstrated a tolerable safety profile with limited single-agent activity. PK/pharmacodynamic readouts of S49076 are encouraging for further investigation of S49076 in combination therapies. Trial registration number ISRCTN00759419.
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