The ß2-adrenergic receptor associates with CXCR4 multimers in human cancer cells.

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

Investigation of the myopic outcomes of the newer intraocular lens power calculation formulas in Korean patients with long eyes.

This study investigated the underlying causes of the myopic outcomes of the optic-based newer formulas (Barrett Universal II, EVO 2.0, Kane, Hoffer-QST and PEARL-DGS) in long Korean eyes with Alcon TFNT intraocular lens (IOL) implantation. Postoperative data from 3100 randomly selected eyes of 3100 patients were analyzed to compare the reference back-calculated effective lens positions (ELPs) based on the Haigis formula using conventional axial length (AL) and Cooke-modified AL (CMAL) with the predicted ELP of each single- and triple-optimized Haigis formula applied to AL- and CMAL. Contrary to the AL-applied Haigis formula, the predicted ELP curve of the CMAL-applied, single-optimized Haigis formula, simulating the methods of the newer formulas, exhibited a significant upward deviation from the back-calculated ELP in long eyes. The relationship between the AL and anterior chamber depth in our long-eyed population differed from that in the base population of the PEARL-DGS formula. The myopic outcomes in long eyes appeared to stem from the substantial overestimation of the postoperative IOL position with AL modification, leading to the implantation of inappropriately higher-powered IOLs. This discrepancy may be attributed to the ethnic differences in ocular biometrics, particularly the relatively smaller anterior segment in East Asian patients with long AL.

Comparison of the formula accuracy for calculating multifocal intraocular lens power: a single center retrospective study in Korean patients.

This study evaluated the accuracy of newer formulas (Barrett Universal II, EVO 2.0, Kane, Hoffer QST, and PEARL-DGS) and the Haigis formula in Korean patients with the Alcon TFNT multifocal intraocular lens. In total, 3100 randomly selected eyes of 3100 patients were retrospectively reviewed. After constant optimization, the standard deviation (SD) of the prediction error was assessed for the entire group, and the root mean square error was compared for short and long axial length (AL) subgroup analysis. The Cooke-modified AL (CMAL) was experimentally applied to the Haigis formula. All the newer formulas performed well, but they did not significantly outperform the Haigis formula. In addition, all the newer formulas exhibited significant myopic outcomes (- 0.23 to - 0.29 diopters) in long eyes. Application of the CMAL to the Haigis formula with single constant optimization produced similar behavior and higher correlation with the newer formulas. The CMAL-applied triple-optimized Haigis formula yielded a substantially smaller SD, even superior to the Barrett and Hoffer QST formulas. The AL modification algorithms such as the CMAL used in newer formulas to cope with optical biometry's overestimation of the AL in long eyes seemed to overcompensate, particularly in the long eyes of the East Asian population.

GPC-100, a novel CXCR4 antagonist, improves in vivo hematopoietic cell mobilization when combined with propranolol.

Autologous Stem Cell Transplant (ASCT) is increasingly used to treat hematological malignancies. A key requisite for ASCT is mobilization of hematopoietic stem cells into peripheral blood, where they are collected by apheresis and stored for later transplantation. However, success is often hindered by poor mobilization due to factors including prior treatments. The combination of G-CSF and GPC-100, a small molecule antagonist of CXCR4, showed potential in a multiple myeloma clinical trial for sufficient and rapid collection of CD34+ stem cells, compared to the historical results from the standards of care, G-CSF alone or G-CSF with plerixafor, also a CXCR4 antagonist. In the present study, we show that GPC-100 has high affinity towards the chemokine receptor CXCR4, and it potently inhibits ß-arrestin recruitment, calcium flux and cell migration mediated by its ligand CXCL12. Proximity Ligation Assay revealed that in native cell systems with endogenous receptor expression, CXCR4 co-localizes with the beta-2 adrenergic receptor (ß2AR). Co-treatment with CXCL12 and the ß2AR agonist epinephrine synergistically increases ß-arrestin recruitment to CXCR4 and calcium flux. This increase is blocked by the co-treatment with GPC-100 and propranolol, a non-selective beta-adrenergic blocker, indicating a functional synergy. In mice, GPC-100 mobilized more white blood cells into peripheral blood compared to plerixafor. GPC-100 induced mobilization was further amplified by propranolol pretreatment and was comparable to mobilization by G-CSF. Addition of propranolol to the G-CSF and GPC-100 combination resulted in greater stem cell mobilization than the G-CSF and plerixafor combination. Together, our studies suggest that the combination of GPC-100 and propranolol is a novel strategy for stem cell mobilization and support the current clinical trial in multiple myeloma registered as NCT05561751 at www.clinicaltrials.gov.

Inactivation of Sirtuin2 protects mice from acetaminophen-induced liver injury: possible involvement of ER stress and S6K1 activation.

Acetaminophen (APAP) overdose can cause hepatotoxicity by inducing mitochondrial damage and subsequent necrosis in hepatocytes. Sirtuin2 (Sirt2) is an NAD+-dependent deacetylase that regulates several biological processes, including hepatic gluconeogenesis, as well as inflammatory pathways. We show that APAP decreases the expression of Sirt2. Moreover, the ablation of Sirt2 attenuates APAP-induced liver injuries, such as oxidative stress and mitochondrial damage in hepatocytes. We found that Sirt2 deficiency alleviates the APAP-mediated endoplasmic reticulum (ER) stress and phosphorylation of the p70 ribosomal S6 kinase 1 (S6K1). Moreover, Sirt2 interacts with and deacetylates S6K1, followed by S6K1 phosphorylation induction. This study elucidates the molecular mechanisms underlying the protective role of Sirt2 inactivation in APAP-induced liver injuries. [BMB Reports 2019; 52(3): 190-195].