Biology subjects

Allen, B. G.

Publications and source records attributed to Allen, B. G..

9 recordsLinked to original sources

bioRxiv2024

Targeting CXCR4 with -Pentixather Significantly Increases Overall Survival in Small Cell Lung Cancer

IntroductionSmall cell lung cancer (SCLC) has a 7% 5-year overall survival. C-X-C chemokine receptor 4 (CXCR4), an attractive target for theranostic agents, is highly expressed in SCLCs, and can be targeted with pentixather using the theranostic pair 212Pb/203Pb. The hypothesis that [212Pb/203Pb]-pentixather can be used safely and effectively for imaging and therapy in SCLC in xenograft models was tested. ResultsSPECT/CT imaging and biodistribution studies of tumor bearing mice injected with [203Pb]-pentixather demonstrated CXCR4-dependent uptake in tumors and accumulation of radioligand in kidneys and livers. Dosimetry calculations estimated [212Pb]-pentixather uptake in tumor and normal tissue. [212Pb]-pentixather treatment (37-111 kBq/g) of SCLC xenografts (DMS273 and H69AR) significantly prolonged survival and delayed tumor growth. When NSG mice grafted with human hCD34+ bone marrow were treated with [212Pb]-pentixather (37-111 kBq/g), significant cytopenias were observed in peripheral blood complete blood counts (CBCs) at 13-18 days post treatment which resolved by day 28-31. Flow cytometry of bone marrow hematopeotic stem cells in these animals at day 28-31 demonstrated a significantly reduced frequency of the human hematopoietic marker CD45 (hCD45+) and reconstitution of the bone marrow with murine CD45+ (mCD45+) lineages. Conclusions[203Pb]-pentixather can be used to image CXCR4 expressing SCLC xenografts and treatment with alpha emitter [212Pb]-pentixather significantly prolongs SCLC xenograft median overall survival. Significantly greater mCD45+ bone marrow repopulation was detected in NSG mice engrafted with human bone marrow 28-31 days following [212Pb]-pentixather treatment, relative to hCD45+ bone marrow.

cancer biology↗

bioRxiv2023

ERK3 Is Involved in Regulating Cardiac Fibroblast Function

ERK3/MAPK6, an atypical MAPK, activates MAP kinase-activated protein kinase (MK)-5 in selected cell types. MK5 haplodeficient mice show reduced hypertrophy and attenuated increase in Col1a1 mRNA in response to increased cardiac afterload. In addition, MK5 deficiency alters cardiac fibroblast function. This study was to determine the effect of reduced ERK3 on cardiac hypertrophy following transverse aortic constriction (TAC) and fibroblast biology. Three wk post-surgery, ERK3, but not ERK4 or p38, was co-immunoprecipitated with MK5 from both sham and TAC heart lysates. The increase in left ventricular mass and myocyte diameter was lower in TAC-ERK3+/- than TAC-ERK3+/+ hearts, whereas ERK3 haploinsufficiency did not alter systolic or diastolic function. Furthermore, the TAC-induced increase in Col1a1 mRNA abundance was diminished in ERK3+/- hearts. ERK3 immunoreactivity was detected in atrial and ventricular fibroblasts but not myocytes. In both quiescent fibroblasts and activated myofibroblasts isolated from adult mouse heart, siRNA-mediated knockdown of ERK3 reduced the TGF-{beta}-induced increase in Col1a1 mRNA. In addition, intracellular type 1 collagen immunoreactivity was reduced following ERK3 depletion in quiescent fibroblasts but not myofibroblasts. Finally, knocking down ERK3 impaired motility in both atrial and ventricular myofibroblasts. These results suggest that ERK3 plays an important role in multiple aspects of cardiac fibroblast biology.

cell biology↗

bioRxiv2023

Auranofin Inhibition of Thioredoxin Reductase in a Preclinical Model of Small Cell Lung Cancer

Thioredoxin Reductase (TrxR) is a key enzyme in hydroperoxide detoxification through peroxiredoxin enzymes and in thiol-mediated redox regulation of cell signaling. Because cancer cells produce increased steady-state levels of reactive oxygen species (ROS; i.e., superoxide and hydrogen peroxide), TrxR is currently being targeted in clinical trials using the anti-rheumatic drug, auranofin (AF). AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the lung atypical (neuroendocrine tumor) NET cell line H727. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, a multi-kinase inhibitor that was shown to decrease intracellular glutathione. The pharmacokinetic and pharmacodynamic properties of AF treatment in a mouse SCLC xenograft model was examined to maximize inhibition of TrxR activity without causing toxicity. AF was administered intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1 to 5 days in mice with DMS273 xenografts. Plasma levels of AF were 10-20 M (determined by mass spectrometry of gold) and the optimal inhibition of TrxR (50 %) was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. When this daily AF treatment was extended for 14 days a significant prolongation of median survival from 19 to 23 days (p=0.04, N=30 controls, 28 AF) was observed without causing changes in animal bodyweight, CBCs, bone marrow toxicity, blood urea nitrogen, or creatinine. These results show that AF is an effective inhibitor of TrxR both in vitro and in vivo in SCLC, capable of sensitizing NETs and SCLC to sorafenib, and supports the hypothesis that AF could be used as an adjuvant therapy with agents known to induce disruptions in thiol metabolism to enhance therapeutic efficacy.

cancer biology↗

bioRxiv2023

MK2 deficiency decreases mortality during the inflammatory phase after myocardial infarction in mice

BackgroundAltering the onset, intensity, or duration of inflammation can impact the recovering hearts structure and function following myocardial infarction (MI). Substrates of MAP kinase-activated protein kinase 2 (MK2) include proteins that regulate the stability of AU-rich transcripts, including those of several pro-inflammatory cytokines. This study was to determine if MK2-deficiency impaired the inflammatory phase of post-MI wound repair. Methods and ResultsMyocardial infarctions were induced by permanent ligation of the left anterior descending coronary artery in 12-week-old male MK2+/+ and MK2-/- littermate mice. Five days post-MI, survival was 100% in MI-MK2-/- (n = 20) and 79% in MI-MK2+/+ mice (n = 29; Mandel-Cox test: P < 0.05). Area at risk and infarct size were similar. Echocardiographic imaging revealed that both systolic and diastolic LV diameters were greater in MI-MK2+/+ than MI-MK2-/- mice. MK2-deficiency did not affect the increase in wall motion score index. Infiltration of neutrophils or monocytes did not differ significantly. Cytokine and chemokine transcripts were quantified in infarcted and non-infarcted LV tissue using qPCR arrays (QIAGEN). Three days post-MI, Ifna2 was increased and Il16 was decreased in infarcted tissue from MK2-/- hearts, compared with infarcted MK2+/+ tissue, whereas in the non-infarcted MK2-/- myocardium Il27 increased and Tnfsf11, Ccl3, and Il1rn were decreased. Five days post-MI, Ctf16 and Il10 increased in infarcted MK2-/- tissue whereas in the non-infarcted MK2-/- myocardium Ccl9, Nodal, and Xcl2 increased and Il15 decreased. ConclusionsThe findings of this study suggest MK2-deficiency is an advantage during the inflammatory phase of cardiac wound repair post-MI. Clinical PerspectiveWhat is new? -The effects of MAP kinase-activated protein kinase 2 (MK2) deficiency on survival, cardiac structure and function, and the inflammatory phase of wound healing following myocardial infarction were assessed using a constitutive, pan MK2-null mouse model. -MK2-deficiency reduced mortality but did not alter area at risk or infarct size post-myocardial infarction. Inflammatory cell infiltration was also unaffected. -MK2-deficiency altered the abundance of several cytokines (increased, decreased) in infarcted and non-infarcted myocardium post-MI. What are the clinical implications? -The initial phase of wound repair post-MI involves inflammation. -The risk of damage to the myocardium and mortality may be reduced by inhibition of MK2 activity during the inflammatory phase of wound healing post-MI.

physiology↗

bioRxiv2023

Tubular Mitochondrial Pyruvate Carrier Disruption Elicits Redox Adaptations that Protect from Acute Kidney Injury

Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.

biochemistry↗

bioRxiv2020

Membrane-tethered peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as allosteric biased ligands

Over the last decade, the urotensinergic system has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases and also cancer. Significant investment toward the development of clinically relevant UT ligands for therapeutic intervention has been made but have met little to no success to date. The UT system, which has yet to be effectively targeted, therefore remains to be therapeutically exploited. The discovery of allosteric sites that allow modulation of receptor activity will increase the searchable chemical space against a disease-relevant target. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics. Therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study UT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of UT, respectively, have been synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and to a lesser extent, IP1 production, stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate hUII- and URP-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue to design allosteric ligands selectively targeting UT signaling that could prove to be useful for the treatment of hUT-associated diseases.

pharmacology and toxicology↗

bioRxiv2020

Expression, subcellular localization, and phosphorylation of MK5 in adult cardiac ventricular fibroblasts

MAP kinase-activated protein kinase-5 (MK5) plays an important role in cardiac fibroblast function. Although p38 MAPK and atypical MAPKs and ERK3 and ERK4 have been identified as activators of MK5, the kinases that activate MK5 remain controversial. Here we examined the expression, subcellular distribution, and regulation of MK5 in cardiac ventricular myofibroblasts and myocytes. The copy numbers for MK5 and ERK4 mRNA were comparable in myocytes and myofibroblasts, whereas that of ERK3 was much higher in myofibroblasts. Interestingly, MK5 and ERK3 immunoreactivity was detected in myofibroblasts but not myocytes whereas ERK4 immunoreactivity was detected in myocytes: treating in myocytes with a proteasome inhibitor or hypertrophic agonists failed to rescue MK5 immunoreactivity. In myofibroblasts, MK5 and ERK3 immunoreactivity was predominantly nuclear and cytosolic, respectively. In serum-starved cardiac myofibroblasts, phosphothreonine-182 MK5 (pT182-MK5) immunoreactivity was predominantly nuclear but increased in intensity and relocated to the cytoplasm in response to serum, sorbitol, angiotensin II, TGF{beta}, or H2O2 and this was prevented by inhibition of p38/{beta}. Phos-tag SDS-PAGE revealed multiple slower migrating bands of MK5 immunoreactivity, indicating phosphorylation of MK5 at multiple sites. Phos-tag PAGE also revealed MK5 phosphorylation was increased with fibroblast activation and in hearts exposed to a chronic increase in afterload. MK5 and ERK3 co-immunoprecipitated and proximity ligation assays revealed ERK3 and MK5 in close proximity in myofibroblast cytoplasmic compartment. Furthermore, p38/{beta} inhibition decreased the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. Finally, deleting MK5 did not reduce the abundance of ERK3 immunoreactivity. These observations suggest that p38 and/or p38{beta} are the primary mediators of T182-MK5 phosphorylation and hence MK5 activation in cardiac myofibroblasts.

biochemistry↗

bioRxiv2020

MK2-deficient mice are bradycardic and display delayed hypertrophic remodelling in response to a chronic increase in afterload

MAP kinase-activated protein kinase-2 (MK2) is protein serine/threonine kinase activated by p38/{beta}. Herein we examined the cardiac phenotype of pan MK2-null (MK2-/-) mice. Survival curves for male MK2+/+ and MK2-/- mice did not differ (Mantel-Cox test, P = 0.580). At 12-weeks of age, MK2-/- mice exhibited normal systolic function along with signs of possible early diastolic dysfunction; however, ageing was not associated with an abnormal reduction in diastolic function. Both R-R interval and P-R segment durations were prolonged in MK2-deficient mice. However, heart rates normalized when isolated hearts were perfused ex vivo in working mode. Ca2+ transients evoked by field stimulation or caffeine were similar in ventricular myocytes from MK2+/+ and MK2-/- mice. MK2-/- mice had lower body temperature and an age-dependent reduction in body weight. mRNA levels of key metabolic genes, including Ppargc1a, Acadm, Lipe, and Ucp3 were increased in hearts from MK2-/- mice. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+-sensitivity to mitochondrial permeability transition pore (mPTP) opening. Finally, the pressure overload-induced increase in heart weight/tibia length and decrease in systolic function were attenuated in MK2-/- mice two weeks, but not eight weeks, after constriction of the transverse aorta. Collectively, these results implicate MK2 in (i) autonomic regulation of heart rate, (ii) cardiac mitochondrial function, and (iii) the early stages of myocardial remodeling in response to chronic pressure overload. Key points summaryThe cardiac characterization of pan MK2-null mice showed: O_LIaltered autonomic regulation of heart rate C_LIO_LIincreased expression of key metabolic genes C_LIO_LIdecreased Ca2+-sensitivity for MPTP opening C_LIO_LIdelayed hypertrophic remodeling in response to increased afterload C_LI

physiology↗

bioRxiv2019

Protein Tyrosine Phosphatase 1B Regulates MicroRNA-208b-Argonaute 2 Association and Thyroid Hormone Responsiveness in Cardiac Hypertrophy

Elevated reactive oxygen species (ROS) production plays an important role in the pathogenesis of several diseases, including cardiac hypertrophy. While the regulation of diverse sources of ROS is well characterized in the heart, the redox-sensitive targets that contribute to redox signaling remain largely undefined. We now report that protein tyrosine phosphatase 1B (PTP1B) is reversibly oxidized and inactivated in hearts undergoing hypertrophy and that gene deletion of PTP1B in mouse hearts cause an hypertrophic phenotype that is critically exacerbated in mice subjected to pressure overload. Furthermore, we show that PTP1B dephosphorylates Tyr393 on argonaute 2, a key component of the RNA-induced silencing complex, and sustains gene silencing in the heart. Our results indicate that PTP1B inactivation and argonaute 2 Tyr393 phosphorylation specifically prevents argonaute 2 from interacting with miR-208b. Phosphorylation and inactivation of argonaute 2 in PTP1B cKO mice revealed a mechanism by which defective miR-208b-mediated repression of thyroid hormone receptor-associated protein 1 (THRAP1/MED13) contributes to thyroid hormone-mediated cardiac hypertrophy. In support of this conclusion, inhibiting the synthesis of triiodothyronine (T3), using propylthiouracil, rescued TAC-induced hypertrophy and improved myocardial contractility and systolic function in PTP1B cKO mice. Together, our data illustrate that PTP1B activity exerts a cardioprotective effect in the heart and that redox signaling is tightly linked to thyroid hormone responsiveness and to microRNA-mediated gene silencing in pathological hypertrophy.

cell biology↗