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.

LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration.

BACKGROUND: G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA1) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA1 heteromer and characterized the unique molecular features and function of this heteromer. METHODS: We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA1. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, ß-arrestin recruitment, ligand binding, and transwell migration assays. RESULTS: We observed that CXCR4 forms heteromers with LPA1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gαi/o activation, and ß-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA1-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gαi/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA1. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA1-selective antagonists enhanced CXCL12-induced Gαi/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA1-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA1 antagonists. CONCLUSIONS: The presence and impact of CXCR4-LPA1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA1 antagonists alone for such conditions. Video Abstract.

Simultaneous activation of CXC chemokine receptor 4 and histamine receptor H1 enhances calcium signaling and cancer cell migration.

C-X-C chemokine receptor 4 (CXCR4) is widely overexpressed in various types of cancer and is involved in several cancer phenotypes including tumor growth, survival, and metastasis. The roles of histamine and histamine receptor H1 (HRH1) in cancer pathogenesis remain controversial. Here, we show that HRH1 is widely expressed in various cancer cell lines and cancer tissues and that coexpression of CXCR4 and HRH1 is associated with poor prognosis in breast cancer. Using bimolecular fluorescence complementation and bioluminescence resonance energy transfer donor saturation assays, we demonstrate that CXCR4 and HRH1 can assemble into a heteromeric complex. Simultaneous activation of CXCR4 and HRH1 synergistically increases calcium flux in MDA-MB-231 cells that endogenously express CXCR4 and HRH1 but not in cells deficient in CXCR4 or HRH1. Costimulation of CXCR4 and HRH1 also significantly enhances CXCL12-induced MDA-MB-231 cell migration, while histamine alone does not induce cell migration. Synergistic effects on calcium flux and cell migration are inhibited by the Gαi inhibitor pertussis toxin and the Gαq inhibitor YM254890, suggesting that the Gαi and Gαq pathways are involved in the synergy. Enhanced calcium signaling and cell migration are also observed in NCI-H23 and HeLa cells, which coexpress CXCR4 and HRH1. Taken together, our findings demonstrate an interplay between CXCR4 and HRH1, and suggest the possibility of the CXCR4-HRH1 heteromer as a potential therapeutic target for anticancer therapy.

AdHTS: a high-throughput system for generating recombinant adenoviruses.

The need for efficient high-throughput gene delivery system for mammalian cells is rapidly increasing with the growing request for functional genomics studies and drug discoveries in various physiologically relevant systems. However, plasmid-based gene delivery has limitations in transfection efficiency and available cell types. Viral vectors have great advantages over plasmid-based vectors, but construction of recombinant viruses remains to be a big hurdle for high-throughput applications. Here we demonstrate a rapid and simple high-throughput system for constructing recombinant adenoviruses which have been used as efficient gene delivery tools in mammalian systems in vitro and in vivo. By combining Gateway-based site-specific recombination with Terminal protein-coupled adenovirus vector, the adenovirus high-throughput system (AdHTS) generates multiple recombinant adenoviruses in 96-well plates simultaneously without the need for additional cloning or recombination in bacteria or mammalian cells. The AdHTS allows rapid and robust cloning and expression of genes in mammalian cells by removing shuttle vector construction, bacterial transformation, or selection and by minimizing effort in plaque isolation. By shortening the time required to convert whole cDNA library into desired viral vector constructs, the AdHTS would greatly facilitate functional genomics and proteomics studies in various mammalian systems.

One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies.

We developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.

Anti-Arthritic and Analgesic Effect of NDI10218, a Standardized Extract of Terminalia chebula, on Arthritis and Pain Model.

The fruit of Terminalia chebula Retzius has been used as a panacea in India and Southeast Asia but its biological activities have not been fully elucidated. Here we report anti-arthritic and analgesic effect of NDI10218, a standardized ethanol extract of Terminalia chebula, on collagen-induced arthritis and acetic acid-induced writhing model, respectively. Arthritis was induced in DBA/1J mice by immunizing bovine type II collagen and mice were treated with NDI10218 daily for 5 weeks after the onset of the disease. NDI10218 reduced the arthritis index and blocked the synovial hyperplasia in a dose-dependent manner. The serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß were significantly reduced in mice treated with NDI10218. Production of the inflammatory IL-17, but not immunosuppressive IL-10, was also inhibited in splenocytes isolated from NDI10218-treated arthritis mice. Administration of NDI10218 markedly decreased the number of T cell subpopulations in the regional lymph nodes of the arthritis mice. Finally, NDI10218 reduced the number of abdominal contractions in acetic acid-induced writhing model, suggesting an analgesic effect of this extract. Taken together, these results suggest that NDI10218 can be a new therapeutic candidate for the treatment of rheuma-toid arthritis.

Foenumoside B from Lysimachia foenum-graecum inhibits adipocyte differentiation and obesity induced by high-fat diet.

We have previously reported anti-obesity effects of Lysimachia foenum-graecum in high-fat diet (HFD)-induced obesity model. Here we isolated a triterpene saponin foenumoside B as an active component of L. foenum-graecum. Foenumoside B blocked the differentiation of 3T3-L1 preadipocytes in a dose-dependent manner with an IC50 of 0.2 µg/ml in adipogenesis assay and suppressed the induction of PPARγ, the master regulator of adipogenesis. Foenumoside B induced the activation of AMP-activated protein kinase (AMPK), and modulated the expression of genes involved in lipid metabolism towards lipid breakdown in differentiated adipocytes. In mouse model, oral administration of foenumoside B (10mg/kg/day for 6 weeks) reduced HFD-induced body weight gain significantly without affecting food intake. Treatment of foenumoside B suppressed lipid accumulation in white adipose tissues and the liver, and lowered blood levels of glucose, triglycerides, ALT, and AST in HFD-induced obese mice. Consistent with the in vitro results, foenumoside B activated AMPK signaling, suppressed the expression of lipogenic genes, and enhanced the expression of lipolytic genes in vivo. Foenumoside B also blocked HFD-induced proinflammatory cytokine production in adipose tissue, suggesting its protective role against insulin resistance. Taken together, these findings demonstrate that foenumoside B represents the anti-obesity effects of L. foenum-graecum, and suggest therapeutic potential of foenumoside B in obesity and obesity-related metabolic diseases.

Anti-obesity effects of Lysimachia foenum-graecum characterized by decreased adipogenesis and regulated lipid metabolism.

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 µg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 µg/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARγ and C/EBPα expression as shown in in vitro and in vivo, and the suppression of PPARγ activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.

Rapid activation of CLOCK by Ca2+-dependent protein kinase C mediates resetting of the mammalian circadian clock.

In mammals, immediate-early transcription of the Period 1 (Per1) gene is crucial for resetting the mammalian circadian clock. Here, we show that CLOCK is a real signalling molecule that mediates the serum-evoked rapid induction of Per1 in fibroblasts through the Ca2+-dependent protein kinase C (PKC) pathway. Stimulation with serum rapidly induced nuclear translocation, heterodimerization and Ser/Thr phosphorylation of CLOCK just before the surge of Per1 transcription. Serum-induced CLOCK phosphorylation was abolished by treatment with PKC inhibitors but not by other kinase inhibitors. Consistently, the interaction between CLOCK and PKC was markedly increased shortly after serum shock, and the Ca2+-dependent PKC isoforms PKCalpha and PKCgamma phosphorylated CLOCK in vitro. Furthermore, phorbol myristic acetate treatment triggered immediate-early transcription of Per1 and also CLOCK phosphorylation, which were blocked by a Ca2+-dependent PKC inhibitor. These findings indicate that CLOCK activation through the Ca2+-dependent PKC pathway might have a substantial role in phase resetting of the circadian clock.